Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions

Microbial Ecology studies aim to describe and assess the behavior and activity of microorganisms in their natural environments (Brock 1966). Nowadays it is clear that the large number of existing microorganisms has surpassed our capabilities to rapidly characterise them by traditional culturing methods. This has resulted in a poor understanding of the structure and composition of microbial communities. As an alternative, microbial communities can be described on the basis of 16S rRNA sequence diversity, without the bias-introducing step of cultivation.In the present thesis a molecular analysis is given of two ecosystems that harbour several uncultured bacteria. The first part of the thesis is focused on the detection and characterisation of Frankia in actinorhizal nodules and soil. Elucidation of the actual diversity within the family Frankiaceae was hampered by the inability to obtain isolates from all known actinorhizal plants. So far, the Nod +/Fix +Frankia symbionts in root nodules of plants from the families Coriariaceae, Datiscaceae, Rosaceae and Rhamnaceae (with exceptions reported by Carú 1993, Carú et al . 1990, and Carrasco et al . 1995) have resisted isolation. Best opportunities to characterise those uncultured endophytes require molecular methods that relay heavily on an easy and efficient technique to extract DNA from the respective actinorhizal nodules. Chapter 2 describes the techniques to isolate DNA from root nodules of different actinorhizal plants such as Casuarina sp, Alnus sp and Ceanothus sp. The procedure has also been successfully applied by Wolters et al . (1997b) in the minuscule ineffective nodules on Alnus glutinosa .Several attempts to characterise the uncultured endophytes from Coriaria sp. and Datisca sp. plants pointed on the one hand, to the presence in those actinorhizae of Frankia -related actinomycetes. This assumption was based mainly on the repeated isolation from those nodules of Nod -/Fix -Frankia -like strains (Hafeez 1983, Mirza et. al . 1994b, c). On the other hand, the effective (Fix +), non-isolated symbionts showed to be phylogenetically closely related (Mirza et al. 1994a), forming a separate lineage within the genus, in spite of the distant geographical distribution of the plants (Nick et al. 1992).The work described in Chapter 3 is focussed on the localisation and phylogenetic position of the nitrogen-fixing Frankia and Nod -/Fix -actinomycetes, both present in root nodules of the Mexican actinorhizal plant Ceanothus caeruleus . Application of the TGGE technique allowed localising the Nod -/Fix -actinomycete in the outer layers of the C. caeruleus nodules. Similar bacteria were also detected in Hippophaë rhamnoides nodules induced with soil inoculum that was collected in the vicinity of the former plant. The fact that a second nodule inhabitant was commonly present in these nodules containing recalcitrant endophytes may allow some speculations about their possible role in the symbiosis. However, it seems worthwhile to apply the same TGGE methodology to other actinorhizal nodules, even to those containing Frankia strains that are easy-to-isolate (i.e. Elaeagnus,Casuarina, Alnus spp.), since the detection of Frankia -related actinomycetes, in addition to the Fix +endophyte, would provide further evidence about the need for their presence. Coincidentally, the Nod -/Fix -isolates from Coriaria, Datisca and Ceanothus are phylogenetically related, pointing again to a certain specificity for their presence in the nodules. This relatedness has also been supported by analysis of low molecular weight RNA (i.e. 5S rRNA and tRNA's) using staircase electrophoresis (Velázquez et al. 1998).The 16S rDNA sequence from the non-isolated Fix +endophyte in C. caeruleus root nodules (Chapter 3), was the first full sequence obtained from a field-collected Ceanothus symbiont. Parsimony and phylogenetic distance analyses grouped it within the Dryas cluster that originally contained only the uncultured endophytes from Dryas , Coriaria and Datisca as proposed by Normand et al. (1996). Benson et al . (1996) redefined this cluster by adding other uncultured endophytes present in Ceanothus griseus (Rhamnaceae), Purshia tridentata and Dryas drummondii (Rosaceae) root nodules. Since the determined partial 16S rDNA sequences were almost identical, they suggested that the Frankia diversity from these actinorhizal plant families might be low. However, Clawson et al. (1998) demonstrated that Frankia isolates obtained from several genera within the Rhamnaceae (i.e. Talguenea , Colletia , Discaria, Retanilla and Trevoa ) were phylogenetically different than those in Ceanothus, grouping in the Elaeagnus cluster. These findings were consistent with morphological differences of the endophytes in planta , since the vesicles found in the Ceanothus symbionts resemble more to those in the Rosaceae, while all the latter host plants in the Rhamnaceae family have endophytes like those in Elaeagnus .The results reported in the first part of the thesis have demonstrated that TGGE and sequence analysis of 16S rDNA provide an accurate picture for the identification of recalcitrant endophytes in root nodules of actinorhizal plants. It has also been demonstrated that besides the N 2 -fixing endophyte, root nodules of C. caeruleus also harbour Frankia -related actinomycetes. Since these have also been observed in other actinorhizae, a further study is needed to understand the possible function of these co-symbionts.The work described in the second part of the thesis was addressing the changes occurring under chlorobenzoate stress in the soil bacterial community and other selected groups of bacteria present in peat soil collected from a natural Alnus glutinosa stand. A combination of culturing and non-culture based approaches was used for the assessment. Among the latter approaches, the possibilities offered by TGGE were exploited in several ways. Profiling of complex communities and subsequent analysis of specific bacterial groups has been one of the major applications of TGGE (Felske et al . 1996). With this approach, major population shifts induced by either 3CBA or 2,5DCB were detected in the uncultured bacterial community (Chapter 4). Although only the former compound was readily metabolised in soil, both xenobiotics promoted similar changes. Several bacterial populations were reduced or suppressed, while few others were enriched in time, as assessed by shifts in the TGGE banding patterns of the total bacterial community.To characterise the soil-enriched bacterial populations, 3CBA-degrading enrichment cultures were obtained and their composition was addressed by TGGE. Further isolation attempts were directed by this means to prove that the isolated strains were indeed the same enriched organisms as detected in soil. One of the enrichment cultures contained two of the soil-enriched bacteria as predominant components. Although isolation was not achieved, both bacteria were identified as belonging to the genus Burkholderia . The bacterial group detected as predominantly enriched in both spiked soils was not present in any of the enrichment cultures, suggesting that the microorganisms belonging to this groups are either unable to degrade 3CBA or not growing under the used culturing conditions. In any case their fitness to the soil conditions imposed by the addition of chlorobenzoates was high, but the mechanisms involved were not elucidated. These bacteria were also identified as Burkholderia by partial 16S rDNA sequence analysis (Chapter 4).The diversity (H) and the equitability (J) indices are important parameters used by ecologists to assess the species richness and the species evenness, respectively, within a community. As the estimation of such indices relies heavily on species definition and individuals enumeration, their application in microbial ecology studies is seldomly possible. Furthermore, assessment of H and J in uncultured bacterial communities must rely on the interpretation of community fingerprints, which should provide means to distinguish between species or operational taxonomic units (OTU), and to estimate their abundance. TGGE community profiling offers both possibilities, and the community changes occurring in the model soil system were evaluated with this original approach (Chapter 5).In addition, H and J indices were also estimated for the fluorescent pseudomonads group, a selected culturable fraction of the bacterial community. OTU recognition was addressed by using TGGE as a ribotype-fingerprinting technique for the isolated fluorescent pseudomonads. Estimation of H and J at the community level without culturing by TGGE profiling, and at the group level by a combination of culturing and TGGE ribotyping should allow to address and compare the population changes occurring, since the target molecule used in both TGGE was the same. Such comparison was only partially possible since most of the bands corresponding to the fluorescent pseudomonads could not be assessed in the community profiles. However, estimation of H and J indices indicated a clear reduction of species richness and individuals abundance in the uncultured community, which was related to the presence of chlorobenzoates in soil. Evaluation of population shifts by indexed values as H and J proved to be a useful means for analysing the community structure in time, and may be used to assess short and long-term responses of a bacterial community to environmental perturbations.Chapter 6 describes the changes in the total frankiae in soil and in the fraction of the population that is able to produce root nodules in Alnus glutinosa seedlings . Culture-independent approaches based on the most probable number concept were used, one in combination with a Frankia -specific PCR detection and another in combination with a plant-nodulation bioassay. After 15 days of incubation in the presence of chlorobenzoates both fractions of the soil Frankia populations were reduced in more than one order of magnitude, while the populations in the unspiked control soil were not affected. The results indicated that 3CBA and 2,5DCB both had a negative effect on the size of the native Frankia population from the used peat forest soil. This negative effect was also evident during in vitro experiments using Frankia strains isolated from Alnus sp. The presence of 1 mM 3CBA in the culture medium, in addition to the normal carbon source, resulted in reduction or suppression of biomass yield.The influence of Alnus glutinosa on the dechlorination of 3CBA by Pseudomonas sp. strain B13 was assessed in hydroponic cultures. It was expected that root exudates could enhance the dechlorination activity of Pseudomonas B13. When the bacteria were incubated in the presence of an alder plant, only a slight increase in the dechlorination rate of 3CBA was registered in comparison to the control without plant. The main observed effect in the alder plants appears to be a protection against 3CBA toxicity, as the alders inoculated with Pseudomonas B13 showed a better survival rate and grew more vigorously than the non-inoculated plants.The toolbox for microbial ecology studies is increasing constantly by means of developing new techniques or by adapting foreign tools into the field, such as indices to evaluate species diversity and eveness. Although the information provided by these community parameters facilitates comparisons and assessment of changes, their suitability to evaluate bacterial communities is still uncomplete. Estimation of diversity indices requires the recognition of bacterial species as discrete units, and this condition is far from real in natural environments. Species-independent approaches to evaluate diversity must be developed, that consider the bacterial diversity as a continuous range of phylogenetically related taxonomic units.In conclusion, the work described in the first part of the thesis strengthen the current phylogenetic division of the Frankiaceae, by adding new evidence supporting two of the already described clusters ( Dryas cluster, Nod -/Fix -cluster). Although the current taxonomic status of the latter cluster must be better evaluated in order to assess its pertinence to the genus Frankia . In addition, the common occurrence of Nod -/Fix -Frankia -like actinomycetes in nodules containing recalcitrant endophytes, mainly from the Dryas cluster, was also demonstrated for Ceanothus actinorhiza. In the second part, the suitability and versatility of molecular tools such as TGGE were demonstrated by their application in community profiling and estimation of bacterial diversity. Community changes occurring in stressed and unstressed soil systems were easily detected and assessed by this means.In addition, specific populations such as the Burkholderia -like bacteria that strongly reacted to the addition of chlorobenzoates to soil were further characterised. Moreover, TGGE was shown to be a fast ribotyping technique that may enable its use in combination with the community profiles to address shifts of specific groups within bacterial communities. It is tempting to suggest that this general approach will be of importance to direct the isolation of hitherto uncultured bacteria from soil.