Polyphasic identification of isolates of Chromobacterium sp . obtained from flooded soil

1 Agronomical Institute of Pernambuco IPA, Genomics Laboratory Recife, Pernambuco, Brazil. Av. Gal San Martin 1371 Bonji 50761-000 RecifePE Brazil. 2 University Federal of Pernambuco UFPE, Department of Biochemistry Recife, Pernambuco, Brazil. 3 University of Pernambuco UPE, Department of Biology Recife, Pernambuco, Brazil. 4 Catholic University of Pernambuco UNICAP, Research Group on Environmental Sciences Recife, Pernambuco, Brazil.


INTRODUCTION
The genus Chromobacterium belongs to the family Neisseriaceae, and the Chromobacterium violaceum species was first proposed by Bergonzini (Boisbaudran, 1882).One of the most notable characteristics of this bacterium is the production of a violet pigment called violacein, when grown under aerobic conditions (Rettori, 1996).C. violaceum is a Gram-negative saprophyte found in soil and water samples of the tropical and subtropical regions of several continents (Durán and Menck, 2001).In Brazil, it is mostly found on the banks of the Rio Negro in the Amazon.The Brazilian National Genome Project Consortium executed the sequencing and analysis of the genome of C. violaceum (Brgene, 2003).Brazil is included among the countries that exhibit an interest in the DNA of this organism, as it is responsible for the production of a variety of secondary metabolites with biotechnological potential (Carraro et al., 2004).
The application of biochemical and molecular techniques in environmental microbiology has facilitated the identification and characterization of new bacterial species.A tool used in determining taxonomic bacterial species is the analysis of fatty acid methyl ester (FAME) (Osterhout et al., 1991).According to Stead et al. (1992), bacterial species can be easily identified using cellular fatty acid profiles, and this method has been found to possess a high level of accuracy.
The sequencing and analysis of the 16S rDNA (Harmsen and Karch, 2004) has been used successfully in diversity studies, and for the identification and classification of various bacteria.The coupling of molecular analysis methods with systematic bacterial and phylogenetic analysis software has identified the relationships between bacterial groups (Turenne et al., 2001;Cohan, 2002).Thus, the present study aimed to identify the isolates of Chromobacterium through their fatty acid profile and by partial sequencing of the 16S rDNA gene.

Microorganisms and culture conditions
The standard strain of C. violaceum ATCC12472 and the other strains that were investigated are listed in Table 1.These isolates were obtained from the collection of the Department of Environmental Sciences at the Catholic University of Pernambuco.The isolates were grown in 250 ml Erlenmeyer flasks containing 50 mL of the LB liquid culture medium (Sambrook and Russel, 2006).The flasks were incubated at 30°C with constant shaking at 150 rpm for 48 h.At the end of the incubation period, the cells were centrifuged at 1,700 x g for 10 min at 4°C.The cell mass was subjected to freeze-drying for the subsequent extraction of fatty acids.

Extraction and methylation of fatty acids
The fatty acids were converted to methyl esters according to the method of Dunlap and Perry (1967).The lyophilized biomass (100 mg) was placed into a screw cap tube, mixed with 2 ml of a solution of boron trifluoride in 14% methanol and 2 mL of benzene and then incubated overnight at 60°C.Following incubation, 2 ml of distilled water was added to the mixture and the tubes were agitated in a vortex for 5 min.The mixture was centrifuged at 1700 xg for 10 min at 40°C.Following centrifugation, the benzene was removed and evaporated with nitrogen, while the fatty acid methyl esters were resuspended in n-hexane and analyzed by gas chromatography (GC).

Gas chromatography
The analysis were performed on a gas chromatograph model Varian CP-3380 coupled to CP-8200 autosampler, capillary column CP SIL 8CB (30 m x 0.25 mm), using helium as the carrier gas.The temperature of the injector and detector (FID) was maintained at 250°C, the temperature "oven" was initially set at 130°C, subsequently increased to 170°C at the rate of 1 to 3°C/min and was finally maintained isothermally at 180°C for 10 min.The fatty acids were identified by comparing the retention times of the peaks of the samples with the standards and the relative amounts of methyl ester accomplished by the composition of cellular fatty acids CFA were calculated by integration of the peak areas.A range of fatty acids standards was obtained from Sigma.

DNA extraction
The Chromobacterium isolates were grown on solid LB medium for 48 h at 30°C.The colonies were transferred to test tubes containing 5 ml of liquid LB medium for 48 h at a temperature of 30°C, kept under constant agitation at 150 rpm and then subjected to DNA extraction.The DNA of the isolates was extracted according to the method described by Weisburg (1991).The material obtained was quantified on a 1.0% agarose gel run at 80 V for 30 min in TBE buffer (Sambrook and Russel, 2006) stained with SYBR gold and visualized and photographed with the 1D Image Analysis Software System (Kodak Digital Science) under UV light.

DNA amplification of the 16S rRNA gene
The 16S rRNA gene present in the isolates was amplified by PCR using the universal primers fD1 and rD1 (Weisburg et al., 1991).The reaction containing 200 mM deoxynucleotidyl, 5 pmoles of each primer, 2 mM MgCl2, 10X PCR buffer, 1.25 U Taq DNA polymerase (Invitrogen) and 40 ng of the template DNA was performed in the Thermal Cycler Gene Amp ® PCR System 9700 (Applied Biosystems), with an initial cycle of denaturation at 94°C for 5 min, then 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 45 s and extension at 72°C for 1 min and 30 s, followed by a final extension at 72°C for 7 min.The amplicons were separated by electrophoresis at 100 V on a 1.0% agarose gel in 0.5 X TBE buffer (Sambrook and Russel, 2006) and stained with Sybr Gold (Invitrogen); fragments with the expected size were visualized under UV light and photographed with the 1D Image Analysis Software system (Kodak Digital Science) under UV light.The 1 Kb Plus DNA Ladder (Invitrogen) was used as a marker.

Sequencing of the 16S rDNA gene fragment
The amplified products were purified with the PCR Kit "GFX TM PCR DNA" (Invitrogen) to remove residual nucleotides, primers and enzyme, thus permitting a sequencing quality reaction.The purified material was sequenced on the MegaBace sequencer (GE Healthcare) using the same primers as those used in the amplification reaction.The sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) program to query the nucleotide similarity to sequences in the GenBank database.

RESULTS
The results of the GC-Gas chromatography analysis identified the presence of fatty acids: lauric acid C12: 0, myristic acid C14: 0, palmitic acid C16: 0, Palmitoleic acid C16: 1 and linolelaidic acid C18: 2. The profiles of the two principal fatty acids that characterize the isolates of Chromobacterium were C16: 0 (palmitic acid) and C16:1 da Silva et al. 691 (palmitoleic).For the fatty acid C12: 0, the lowest amount was produced in the isolate UCP1035 (4.2%) while the highest amount was produced in the isolate UCP1463 (8.5%); however, this fatty acid was not detected in the isolate UCP1471.In all the bacterial species, only a small percentage of fatty acids are freely expressed and can be easily isolated by extracting the cells with the appropriate organic solvent.For the fatty acid C14:0 expressed in isolates of Chromobacterium, the lowest amount was retrieved in the isolate UCP1462 (5.3%), while the highest amount was in UCP1468 (13.4%).In contrast, the highest concentration of the fatty acid C18: 2 was detected in the isolates UCP1462, UCP1465, UCP1466 and UCP1471 at 24.0, 22.0, 20.0 and 22.0%, respectively (Figures 1 and 2).A reduction in the concentration of C18: 2 was observed in the UCP1461 and UCP1467 isolates, which had relatively low concentrations (3.0%) of the fatty acid; this result confirms the C18: 2 content (2.0%) observed in the standard strain ATCC12472.
A comparison of the 16S rDNA sequences of isolates in our study with sequences from the GenBank (NCBI) confirmed the entire genus Chromobacterium.The phylogenetic tree (Figure 3) of these sequences constructed using the Tamura-Nei model for nucleotides and pairwise deletion with 1000 bootstrap replicates showed that all the isolates (UCP1461, UCP1462, UCP1463, UCP1464, UCP1468, UCP1470 and UCP1471) belong to the same group.The 16S rRNA gene of the Chromobacterium isolates showed fragments of sizes ranging from 848-942 bp.There was a high degree of sequence identity in the 16S rDNA gene from all isolates of the Chromobacterium species (Table 2); the sequences of 12 of the isolates exhibited 98% identity with the sequence of the 16S rDNA of C. violaceum ATCC 12472.The highest identity between the isolates was among the species C. violaceum and C. pseudoviolaceum, which showed a 100% identity between the isolates (UCP 1462, UCP1463, UCP 1469 and 1470), while the isolate UCP1489 showed an identity with C. subtsugae (100%).The tree based on these sequences confirmed the close phylogenetic relationship of the isolates of Chromobacterium (UCP1461, UCP1462, UCP1463, UCP1464, UCP1468, UCP1470 and UCP1471) with C. violaceum and C. pseudoviolaceum (Figure 2).Furthermore, the resulting tree revealed two clusters: cluster 1 grouped all the isolates from the Amazon region and the Ceara and was further divided into four sub-clusters.The first sub-cluster included seven isolates of Chromobacterium, sub-cluster 2 included isolated UCP1465 and the species C. violaceum and C. pseudoviolaceum, and finally the subclusters 3 and 4 were monophyletic with the branch isolates UCP1035 and UCP1466, respectively.Cluster 2 had 2 sub-clusters: the first sub-cluster with isolated UCP1489 with the species C. subtsugae and C. piscinae confirmed that the isolate did not group together based on their origin and the second sub-cluster with the   species C. aquaticum and C. heamolyticum.The results obtained with the 16S rRNA can be corroborated by the similarities observed with sequences deposited in the database, which can be assessed following this type of analysis.The data obtained in each tree species, separately with Chromobacterium, only isolated UCP1462, UCP1469 and UCP1470 and showed 100% identity with the species C. violaceum ATCC12472 and C. pseudoviolaceum LMG3953T (Table 2 and Figures 3,  4 and 5).

DISCUSSION
The highest levels of fatty acids were found in the standard strain ATCC12472 were C16: 0 (palmitic acid) and C16: 1 (palmitoleic acid).Additionally, the fatty acid composition that we identified corroborates previous findings by other groups in bacteria (Oliver and Colwell, 1973;Kamimura et al., 1992).For most of the isolates that were analyzed, the highest observed concentration among the saturated fatty acids was that of palmitic acid (C16: 0), except for the UCP1466, UCP1489 and UCP1035 which exhibited higher amounts of palmitoleic acid (C16: 1) at 40.7, 38.0 and 40.0%, respectively.Delong and Yayanos (1985), working with Vibrio, reported the presence of fatty acids C16: 1 and C18: 1.The major problem involved in the study of bacterial fatty acids is identifying an extraction procedure in which all the fatty acids that act on cells are well characterized because the number of cells required to obtain an optimum extraction varies with the bacterial species (Pacheco, 2009) The similarity between the isolated strain and the UCP1469 standard C. violaceum ATCC 12472 was 100%.Moreover, the isolates collected in the Amazonas grouped separately from those collected in Ceará and Pernambuco States.The complete sequence of the 16S rDNA can stratify the position of the bacteria at the genus level and, in some cases, at the species level as well (Garrity and Holt, 2001).Thus, the 16S rDNA must be used, preferably in studies of taxonomy and phylogeny, with an effort to deposit a large number of these sequences related to other ribosomal genes.Sequencing of the 16S rDNA gene has been frequently used as a molecular marker, making this sequence useful in the genomic fingerprinting of bacteria (Lima-Bittencourt et al., 2011) Corroborating the results of the authors cited in the  Stackebrandt and Goebel (1994) proposed a similarity in the sequences of ≥97% for delimiting bacterial species.However, the 16S rRNA is considered a neutral marker, used to distinguish bacteria to the species level according to their evolutionary history of common ancestry, although with some limitations (Oren, 2004).The characterization of bacteria can be effectively performed by identifying and characterizing the sequence information obtained from the 16S rDNA of several samples of Chromobacterium sp., as previously accomplished by Lima-Bittencourt et al. (2011).
A similar result was observed by Hungria et al. (2005) on analyzing the 16S rDNA sequences of C. violaceum to identify the genetic diversity among isolates collected from UFAM in Rio Negro -Brazilian Amazon; the study identified two new groups of species of C. violaceum as has been previously described.We know that microorganism play an essential role in stabilizing the ecosystem through recycling and degradation of pollutant compounds.The C. violaceum has a great biotechnological potential and new techniques for bioprospecting and biodiversity can be an important source of genetic resources in the pharmaceutical, cosmetic and food industry; additionally, these new species of C. violaceum can significantly contribute to the discovery of new compounds and metabolites.The saturated fatty acid profile (specifically palmitoleic (C16: 1) exhibited by the isolates UCP1035, UCP1489 and UCP1466 was different from that of the other isolates studied.The confirmation of the phylogeny of the 16S rDNA gene revealed that most isolates of Chromobacterium were grouped in cluster 1; however, UCP1035 and UCP1466 formed monophyletic branches.Grouped in cluster 2 were UCP1489 and C. piscinae alone.The isolates UCP1462, UCP1469 and UCP1470 shared 100% identity with the strains C. violaceum and C. pseudoviolaceum.
Fatty acids have been used as a tool to study the polyphasic, which is a chemotaxonomic method easier to analyze a large number of isolates in a short period of time.Concomitantly, we also used the sequencing technique of 16S rDNA gene which is more complex and aimed to give more credibility to the obtained results.

Figure 4 .Figure 5 .
Figure 4. Identity maximum of 16S rDNA sequences among isolates NR_074222.1 C. violaceum ATCC 12472 with the isolates studied in this work using the method of constructing phylogenetic Neighbor Joining with maximum parsimony analysis 0.75

Table 1 .
List of Chromobacterium isolates, origin and sequence of 16S rDNA.