Katarina Björklöf

High frequency of conjugation versus plasmid segregation of RP1 in epiphytic Pseudomonas syringae populations

The maintenance and transfer of the broad host-range plasmid RP1 in epiphytically growing populations of Pseudomonas syringae was monitored in the phyllosphere of bush bean (Phaseolus vulgaris). When foliage was inoculated with plasmid-containing bacteria, the plasmid was lost from the majority of the cells within 2 d but was stably maintained in 0.8% of the population. A high frequency of conjugation between added donors and recipients was observed under high humidity conditions. In 1 d, the number of transconjugants rose to 10(-1) of the donors and the proportional level of transconjugants continued to increase until 3 d after inoculation. Under these conditions the proportion of plasmid-containing bacteria stabilized at about 0.8% of the total population. The conjugation rate appeared to be in equilibrium with plasmid loss and the slower growth of the plasmid-carrying cells. A factor that influenced the high conjugation frequency observed was the available nutrients provided by the leaf and also, to a lesser extent, the leaf surface itself. Transfer of the plasmid from added donors to indigenous bacteria was also studied, using a donor-specific bacteriophage for counterselection of the donor. Transfer was observed to 10 different species of Gram-negative epiphytically growing bacteria. The bean leaf surface appears to function as a hotspot at least for intraspecific transfer of plasmids in high humidity. The frequency of transfer was higher than in soil or in rhizosphere habitats. This is likely to be the result of an environment that is nutritionally rich in combination with a limited colonizable surface area which permits close contact between the bacterial cells.

Biomarkers for monitoring efficacy of bioremediation by microbial inoculants.

Bioaugmentation of contaminated sites with microbes that are adapted or genetically engineered for degradation of specific toxic compounds is an area that is currently being explored as a clean-up option. Biomarkers have been developed to track the survival and efficacy of specific bacteria that are used as inocula for bioremediation of contaminated soil. Examples of biomarkers include the luc gene, encoding firefly luciferase and the gfp gene, encoding the green fluorescent protein (GFP). The luc gene was used to tag different bacteria used for bioremediation of gasoline or chlorophenols. The bacteria were monitored on the basis of luciferase activity in cell extracts from soil. The gfp gene was also used to monitor bacteria during degradation of chlorophenol in soil, based on fluorescence of the GFP protein. Other biomarkers can also be used for monitoring of microbial inocula used for bioaugmentation of contaminated sites. The choice of biomarker and monitoring system depends on the particular site, bacterial strain and sensitivity and specificity of detection required.

Monitoring bioremediation of atrazine in soil microcosms using molecular tools

Molecular tools in microbial community analysis give access to information on catabolic potential and diversity of microbes. Applied in bioremediation, they could provide a new dimension to improve pollution control. This concept has been demonstrated in the study using atrazine as model pollutant. Bioremediation of the herbicide, atrazine, was analyzed in microcosm studies by bioaugmentation, biostimulation and natural attenuation. Genes from the atrazine degrading pathway atzA/B/C/D/E/F, trzN, and trzD were monitored during the course of treatment and results demonstrated variation in atzC, trzD and trzN genes with time. Change in copy number of trzN gene under different treatment processes was demonstrated by real-time PCR. The amplified trzN gene was cloned and sequence data showed homology to genes reported in Arthrobacter and Nocardioides. Results demonstrate that specific target genes can be monitored, quantified and correlated to degradation analysis which would help in predicting the outcome of any bioremediation strategy.

Pili of Pseudomonas syringae pathovar syringae enhance initiation of bacterial epiphytic colonization of bean

SUMMARY: Pseudomonas syringae pathovar syringae R32 expresses phage-|6-specific pili that function as adhesins anchoring bacterial cells to the surface of plants. Phage-resistant piliated and non-piliated mutants were compared to the wild-type strain with regards to pellicle formation and performance during different phases of epiphytic colonization of bush bean. The degree of piliation did not affect the ability of the strains to grow on the undisturbed plant surface. The presence of pili did, however, correlate strongly with the efficiency of the strains to initiate colonization from a liquid inoculation suspension if unadsorbed bacteria were removed by rinsing. During early colonization, wild-type bacteria became virtually resistant to displacement by rinsing within 1 d after inoculation, whereas non-piliated mutant bacteria became only partly resistant within 3 d. Piliated cells formed a pellicle on the surface of stationary liquid cultures whereas non-piliated mutant strains did not. A mechanism similar to pellicle formation may be functional on the plant surface, explaining in part the difference in resistance to removal by rinsing.

Monitored Natural Attenuation

Monitored natural attenuation (MNA) is an in situ remediation technology that relies on naturally occurring and demonstrable processes in soil and groundwater which reduce the mass and concentration of the contaminants. Natural attenuation (NA) involves both aerobic and anaerobic degradation of the contaminants due to the fact that oxygen is used up near the core of the contaminant plume. The aerobic and anaerobic microbial processes can be assessed by microbial activity measurements and molecular biology methods in combination with chemical analyses. The sampling and knowledge on the site conditions are of major importance for the linkage of the results obtained to the conditions in situ. Rates obtained from activity measurements can, with certain limitations, be used in modeling of the fate of contaminants whereas most molecular methods mainly give qualitative information on the microbial community and gene abundances. However, molecular biology methods are fast and describe the in situ communities and avoid the biases inherent to activity assays requiring laboratory incubations.

Presence of Actinobacterial and Fungal Communities in Clean and Petroleum Hydrocarbon Contaminated Subsurface Soil

Relatively little is known about the microbial communities adapted to soil environments contaminated with aged complex hydrocarbon mixtures, especially in the subsurface soil layers. In this work we studied the microbial communities in two different soil profiles down to the depth of 7 m which originated from a 30-year-old site contaminated with petroleum hydrocarbons (PHCs) and from a clean site next to the contaminated site. The concentration of oxygen in the contaminated soil profile was strongly reduced in soil layers below 1 m depth but not in the clean soil profile. Total microbial biomass and community composition was analyzed by phospholipid fatty acid (PLFA) measurements. The diversity of fungi and actinobacteria was investigated more in detail by construction of rDNA-based clone libraries. The results revealed that there was a significant and diverse microbial community in subsoils at depth below 2 m, also in conditions where oxygen was limiting. The diversity of actinobacteria was different in the two soil profiles; the contaminated soil profile was dominated by Mycobacterium -related sequences whereas sequences from the clean soil samples were related to other, generally uncultured organisms, some of which may represent two new subclasses of actinobacteria. One dominating fungal sequence which matched with the ascomycotes Acremonium sp. and Paecilomyces sp. was identified both in clean and in contaminated soil profiles. Thus, although the relative amounts of fungi and actinobacteria in these microbial communities were highest in the upper soil layers, many representatives from these groups were found in hydrocarbon contaminated subsoils even under oxygen limited conditions.