Polymorphic DNA fi ngerprinting patterns in distinct Chromobacterium violaceum strains using ERIC-PCR analysis

[a] Ph.D. in Science (Nuclear Energy in Agriculture), Universidade de São Paulo (USP), associate professor I at the Universidade Federal do Vale do São Francisco (UNIVASF), Petrolina, PE Brasil, e-mail: eyre.gabriel@gmail.com [b] Pharmacist, Pontifícia Universidade Católica do Paraná (PUCPR), Curitiba, PR Brasil. [c] Biologist, Pontifícia Universidade Católica do Paraná (PUCPR), Curitiba, PR Brasil. [d] Ph.D. in Animal Science, University of Nebraska, professor at the Pontifícia Universidade Católica do Paraná (PUCPR), São José dos Pinhais, PR Brasil, e-mail: h.madeira@pucpr.br [e] Biomedical microbiologist, Universidade Católica do Paraná (PUCPR), Curitiba, PR Brasil, e-mail: kari.lidani@gmail.com


Introduction
The Chromobacterium violaceum is a free-living bacterium commonly found in the soil and in the water from tropical and subtropical regions around the world.The violacein represents the major pigment produced for this microorganism and several studies have intensively investigated its activity as bactericide, tumoricide and anti-viral agent (1,2).Recently, the complete genome sequence of C. violaceum had provided relevant clues about its physiological adaptation to tropical soils and aquatic habitats, its capacities as an opportunistic pathogen, and its potential biotechnological and pharmaceutical applications (3).Nevertheless, few reports in literature have evaluated the degree of biodiversity among distinct strains of C. violaceum; although this bacterium represents an important ecosystem component of Amazon region.
Under the past decade, arbitrary amplifi cation of polymorphic DNA sequences, such as random amplifi ed polymorphic DNA (RAPD), has extensively been reported as a method for the genetic characterization of microorganisms (4).As reactions and the highly polymorphic DNA fi ngerprinting profi le observed for each bacterial strain, these fi ndings create real possibilities for the detection of C. violaceum strains in microbial mixed cultures through ERIC-PCR assays.
Polymorphic DNA fi ngerprinting patterns in distinct Chromobacterium violaceum strains using ERIC-PCR analysis 131 reactions, was originally designed from the central inverted repeat of the ERIC consensus sequence located in extragenic regions of various enterobacteria (5, 6).Under specifi c experimental conditions, the PCRs were performed from 250 ng of genomic DNA template, 0.5 μM primer ERICII, 2.5 mM MgCl 2 , 0.4 mM dNTPs and 0.2 u Taq DNA polymerase (Invitrogen) in PCR buffer 1x concentrated (50 mM KCl, 20 mM Tris-HCl, pH 8.4), in a fi nal volume at 25μL.After denaturation for 30 sec at 95 ºC, the samples were submitted to annealing for 1 min at 50 ºC and extension for 5 min at 72 ºC, in a total of 40 cycles.The ERIC-PCR products were separated on a 1.5% agarose gels prepared with TBE 1x (90 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 100 mM boric acid), stained with ethidium bromide and photographed by using digital camera (Kodak ™ , model EDAS 290).All assays described above were carried out in triplicate in order to confi rm the reproducibility of results generated.

Data analysis of ERIC-PCR products
Clustering analysis of the ERIC-PCR products and genetic similarity phenograms among the strains were established by using the coeffi cient of Sorensen-Dice (SD) and the UPGMA (unweighted pair-group method, with arithmetic mean) algorithm in the NTSYS-pc version 2.0 (10) computer program.Genotypic diversity and the Shannon gene diversity indexes among the bacterial strains were calculated using the population genetics package POPGENE 1.31 (11).

Results and discussion
In the present study, we characterized genetically distinct C. violaceum strains based on their polymorphic DNA fi ngerprinting patterns.The size and the number of amplifi ed bands were established for each strain by visualizing of the ERIC-PCR products on agarose gels, as observed in Figure 1.The eletrophoretical profi le generated from ERIC-PCR products in the ATCC12472 and CBMA.1 strains yielded multiple distinct DNA fragments of sizes ranging from 100 to 3,000 bp and the number of amplifi ed bands was corresponding to 17 bands per individual.Alternatively, the C. violaceum 27.1 and 07.1 strains also presented a Within this context, the aim of the present study was to characterize genetically distinct C. violaceum strains from their polymorphic DNA fi ngerprinting patterns by using ERIC-PCR analysis.

Strains and growth conditions
Four C. violaceum strains designed originally as ATCC12472, CBMA-1, 07.1 and 27.1 were investigated in the present study.The C. violaceum strain ATCC12472 was obtained from the Fundação Tropical de Pesquisas André Tosello, Campinas (São Paulo, Brazil), whereas the strains CBMA-1, 07.1 and 27.1 were assigned from the Embrapa Soja, Londrina (Paraná, Brazil).These strains were plated into nutrient agar containing 250 μg/mL of ampicilin for 16 hour incubation at 30 °C.

Genomic DNA extraction and ERIC-PCR assays
Saturated cultures from colonies isolated of each C. violaceum strain, have grown aerobically in 6 mL of nutrient broth (NB) containing 250 μg/mL of ampicilin for 16 hour incubation at 30 ºC, and were sampled for extraction of the total genomic DNA, as described by Sambrook and Russell (9).Pellet of cells was lysated in buffer containing 50 mM glucose, 25 mM Tris-HCl, pH 7.5, 10 mM EDTA, pH 8.0 and 1% SDS.After incubation for 1 hour at 55 ºC, samples were submitted to extraction with phenol:chloroform (1:1) and centrifuged at 12,000 xg for 20 minutes.Approximately 800 μL of aqueous phases was precipitated with two volumes of absolute ethanol by incubating for 1 hour at room temperature.Genomic DNA pellet was resuspended in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0) containing 50 μg/mL of RNase (Promega) and incubated at 37 ºC for 1 hour for cleavage of contaminating RNA molecules.The concentration of genomic DNA samples was estimated in spectrophotometer (Beckman, model DU530) and the integrity of these samples was visualized on agarose gels.
The primer ERICII 5'AAGTAAGTGA CTGGGGTGAGCG3' used in the amplifi cation Repetitive element sequences, usually situated in the intergenic space of prokaryotic genomes, as the ERIC sequences, are often highly conserved among dissimilar bacteria, and they have successfully been used to evaluate the bacterial diversity (8, 14).Based on sharpness of amplifi ed DNA bands, four main groups including individuals belonged to specifi c strains were defi ned by clustering analysis of ERIC-PCR products, in which similarity coeffi cients varied from 0.57 to 1 (Figure 2 and Table 1).Except by one unique exemplar belonged to strain 07.1, our results showed typical polymorphic amplifi cation patterns for each strain tested (Figure 2).As shown in Table I, the inter-strain genetic distance and identity calculations based on Nei (15) revealed that the largest distance was observed between C. violaceum ATCC12472 and 07.1 strains (0.6414) and that the greatest identity between those identifi ed as C. violaceum ATCC12472 and CBM strains (0.7622), confi rming the pattern generated in the similarity phenogram (Figure 2).Under our analysis, the genetic diversity estimated from the percentage of polymorphic loci at the C. violaceum ATCC12472 and 27.1 strains was 16%, whereas the 07.1 and CBMA-1 strains presented values corresponding to 36% and 12%, respectively.Based on genic variation statistics for all loci, the data concerning the number and the percentage of polymorphic loci for four strains was 20 and 80%, respectively.typical DNA amplifi cation pattern in comparison to other strains analyzed (Figure 1).In these strains, it was observed that the size of ERIC-PCR products ranged of 150 to 3,000 bp, whereas the number of amplifi ed bands was corresponding to 15 and 16 for 27.1 and 07.1 strains, respectively.
Although some bands in common (PCR products of analogous mobility) are identifi ed between intra and inter-individual strains, the ERIC-PCR analysis revealed clearly distinct DNA amplification profi les from all strains analyzed, as shown in Figure 1.The most common disadvantage pointed into arbitrary amplifi cation protocols concerning the reproducibility during performance of assays (4, 12).Therefore, results presented in this study provide strong evidences of the accurate repeatability in eletrophoretical patterns generated from amplifi cation reactions (Figure 1), suggesting that the polymorphic DNA fi ngerprinting method used in the present study is extremely sensible to detect minor differences between different strains at the same bacterial strains, the same result documented for Escherichia coli strains (13).These fi ndings reinforce the use of ERIC-PCR technique as a powerful tool in the molecular genetic analysis by discriminating individual isolates and identifying of the bacterial strains and for bacterial taxonomy, as well as it may reveal relevant phylogenetical relationships.The C. violaceum strains have been extensively characterized concerning their microbiological properties (1, 2), although few reports in literature have described the polymorphism DNA pattern in these strains.In a recent study, Hungria et al. (17) showed a high level of genetic diversity in isolates of C. violaceum from the waters of river Negro in Brazil.Such fi ndings showed that Brazilian isolates would fi t into at least two new clusters besides C. violaceum species by 16S and 23S ribosomal DNA sequence analysis.Alternatively, the genotyping of C. violaceum isolates was established by recA PCR-RFLP analysis, generating in a rapid nucleotide sequencebased approach to specifi cally identify and classify this bacterium at level of genospecies (18).The results presented in this study revealed that ERIC-like sequences are present at a highly polymorphic amplifi cation pattern in the genomes of gram-negative C. violaceum bacteria, supporting and extending the fi ndings described by Versalovic et al. (13) and de Bruijn (8).In a preliminary study, ERIC-PCR has been used for genetic differentiation of Enterobacter cloacae isolates (19), being evaluated as highly discriminative method for such application.The development of more sensible methods to discriminate bacterial communities is a crescent needed in the area of microbial ecology and in this respect our fi ndings create real possibilities for the detection of C. violaceum strains in microbial mixed cultures through ERIC-PCR procedure.On the other hand, these results also provide new insights into the better understanding of the high biodiversity found in these bacterial strains.