Molecular Methods for Biodiversity Analysis of PSB

Alvaro Peix¹and Eustoquio Martínez-Molina²

¹Departamento de Producción Vegetal. IRNA-CSIC. Salamanca. SPAIN.

²Departamento de Microbiología y Genética. Universidad de Salamanca. SPAIN

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Although the phosphate solubilizing potential is not a very common characteristic among microorganisms, at present phosphate solubilizers belonging to diverse groups of microorganisms, especially bacteria, are known.

From the bacteria, most of the known PS species belong to pseudomonads, bacilli and rhizobia groups, although there are other Genera such as Acinetobacter or Enterobacter which have been described as phosphate solubilizers.

With the new phylogenetic classification of bacteria, the outlines of which are recorded in Bergey’s Manual (http://www.cme.msu.edu/bergeys/), the pseudomonads group has been divided into diverse Genera able to solubilize phosphate in plates, and these Genera have not only been separated from the original Genus but are also to be found in different subclasses of Proteobacteria. For example, Burkholderia (Pseudomonas) cepacia has been included in b–Proteobacteria. The same has occurred with rhizobia which have been divided into several families. In this way, for example Mesorhizobium (Rhizobium) mediterraneum has been separated from family Rhizobiaceae to family Phyllobacteriaceae. Within bacilli, Paenibacillus (Bacillus) polymyxa has been included in the new family Paenibacillaceae. Within eukaryotes, although they have not been widely studied, the phosphate solubilization by fungi of the Genera Penicillium and Aspergillus (Hermosa, pers. comm..), as well as by some yeast as Yarrowia lypolytica is known. Therefore, it is necessary to have techniques which allow the analysis of biodiversity of PS populations and the identification of the different isolates.

Nowadays, techniques based on nucleic acids electrophoresis are the most commonly used for the analysis of microbial populations. Of these, the most used is DNA, although certain molecules of RNA have been proposed as molecular fingerprints of microorganisms which may be applied to the study of microbial populations (Höfle, 1988). These molecules have been named low molecular weight RNA (LMW RNA) and they include the 5S rRNA and tRNA in prokaryotes (both in archaea and bacteria). In eukaryotes, besides these molecules, the 5.8S rRNA forms part of the LMW RNA profiles. This differentiating characteristic between prokaryotes and eukaryotes is very useful when analyzing isolates from complex populations, since in the eukaryotes there is always one molecule more (5.8S rRNA) which, besides, has different sizes for each eukaryotic genus, when a voltage gradient electrophoresis is used for separating these molecules, when they have similar sizes (Cruz-Sánchez et al., 1997).

The results obtained up to now have allowed researchers to establish that LMW RNA molecules separated by staircase electrophoresis are molecular images of each microbial species both in the case of eukaryotes and prokaryotes (Velázquez et al., 2001). According to the results obtained so far, each prokaryote genus has a characteristic 5S rRNA zone and each eukaryote genus has a different combined zone of 5S and 5.8S rRNA. On the other hand tRNA profiles, both class 1 and class 2, are different in each species of prokaryotes and eukaryotes belonging either to the same or different genus. It has also been demonstrated that LMW RNA profiles of all the strains belonging to the same species are identical (Velázquez et al., 2001). Hence LMW RNA profiles are an excellent technique for analyzing populations diversity as they can be applied to a large number of strains in a quick, easy and precise way without intra-specific variations.

In spite of all this, for the time being, DNA is far more widely used in taxonomy and biodiversity studies, especially due to the fact that PCR is more and more available and affordable and analyses can be carried out in almost every laboratory.

There are numerous PCR-based techniques that can be applied to the study of biodiversity. Among them, some are based on sequencing of reaction products but techniques of this kind are more useful in taxonomy than in studies of large populations, as sequencing is still too complex a method to be applied to many strains at the same time. For this reason, other techniques have been developed in order to obtain the same results without the need of gene sequencing. Such techniques are known as DGGE and TGGE which are based on amplification of a G+C hyper-variable content zone which allows the separation of bands of the same size (generally in 16S rDNA) based on their G+C content obtained by means of variation of temperature during electrophoresis in polyacrylamide gels through a time or spatial gradient (Muyzer, 1999).

Other techniques are based on the amplification of certain molecules followed by digestion with several restriction enzymes, obtaining RFLP profiles which can be mathematically analysed to establish clusters (ARDRA). Nevertheless, the usefulness of RFLP in taxonomy has not been well established because are dependent on the type of molecule and restriction enzymes used. While DGGE and TGGE have been applied to prokaryotes, for the time being the RFLP profiling has also been widely applied to eukaryotes and therefore can be a very useful technique in biodiversity studies. However, it has the disadvantage that the taxonomic level which this technique can differentiate is not well established since, depending on the zone where restriction is applied it may show differences at genus level in some groups, at species level in others and even at strain level in the case of ITS1 and ITS2 in fungi. ITS1 and ITS2 profiles are used a lot in eukaryotes because they are very variable, especially in different genera allowing the establishment of differences directly according to the size of the bands amplified with the same primers.

Finally, other techniques are based on direct electrophoresis of amplified fragments by PCR. In these cases the difference in the various procedures usually depends on the type of primer used. Sometimes small primers and low annealing temperatures are used; these lead to random amplifications within the microbial genome thus obtaining RAPD profiles. Intra-specific variations have been described in the RAPD both in bacteria and in fungi and therefore this technique is of great use in biodiversity studies, but at an infra-specific level. In other cases one or two primers designed on a basis of repeated sequences in the genome of the microorganisms are used. This is the case of Box-PCR which uses one single primer and of ERIC-PCR and rep-PCR which employ two primers in order to obtain DNA profiles. In all these cases intra-specific variations have also been observed, so the usefulness of these techniques is very similar to that of RAPD.

Recently a new procedure has been described (Rivas et al., 2001) for obtaining DNA profiles that are effective in biodiversity and taxonomy since the variations observed are established at subspecies level within the same species. This method is based on the use of the two universal primers used to make a complete amplification of the 16S rDNA increasing its concentration about ten times and applying an annealing temperature of 50º C-55ºC.

Some techniques for analyzing biodiversity are based on extra-chromosomal material which, in some cases, involves a high percentage of the genetic material of the bacteria, above all in the case of fast-growing rhizobia. The plasmid profiles analysed by horizontal electrophoresis in agarose gels are a useful tool for the analysis of population diversity that is usually applied in the case of rhizobia.

As well as nucleic acids, proteins are also normally used in microbial biodiversity studies although they have the disadvantage of the fact that the culture conditions always have to be the same in order to avoid the existence of variations in the electrophoretic profiles (SDS-PAGE) which have their origin in differences in culture media composition or in incubation conditions (temperature, time, etc.). The same can be said of the LPS profiles which are useful in biodiversity studies whenever they are analysed under the same culture conditions in all the strains.

Therefore, at present a wide range of molecular tools is available for biodiversity analysis that facilitate the study at different taxonomic levels which can be applied to large populations in order to establish clusters of different microorganisms which is of great help in the later identification of representative strains from the different groups obtained.

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