The phylogeny of bacteria has received very attention and discussion on microbial ecology. Molecular structures and genomics sequences have been used to infer phylogenetic hypothesis among different organisms    . On the last decades, morphological characteristics ceased to be the main default to compare microorganisms and the improvement of molecular techniques contributed to new data that could help on the classification of organisms   . Woese et al. (1990)  showed in a study based on small subunit ribosomal RNA (SSU rRNA 16S) that the life in our planet should be divided into three primary groups of organisms: Bacteria, Eukaria and Archaea. However, different topologies are generated when phylogenetic hypotheses are made with other genes.
In Bacteria domain the different topologies can be result of horizontal gene transfers (HGTs) that occur frequently in these organisms   . Nakamura and his collaborators (2004)  revealed that considerable proportions of most bacterial genomes consist of horizontally acquired genes and the HGT provides basis for quantitatively understanding the evolution of the prokaryotic genome. Many events of HGT occur likely to cause deleterious effects in the chromosome of the host, whereas other acquisitions might be effectively neutral. However, the HGT that confers a selective advantage to the bacterial recipient has potential to spread rapidly within a bacterial population as well as mobile genetic elements that encode their own transfer and maintenance functions  . Despite of genes of the central cellular machinery such as replication, maintenance, transcription and translation which tend not to spread within a population, some housekeeping genes were reported in integrative and conjugative elements  .
Petroleum hydrocarbons enter the environment directly of accidents, spills during transportation and extraction, leakage from waste disposal or storage site and industrial facilities  . The entry of petroleum in marine environments receives attention because it causes many problems. Polycyclic aromatic hydrocarbons (PAH) are present as natural constituents in crude oil and are highly recalcitrant. The size of the hydrocarbon molecule is inversely proportional to its volatility, water solution and possibility to be degraded by microorganisms   . These compounds perform chronic effects on the local biota, like endocrine deregulation, stress, affect the competition for food, and are carcinogenic and other physiological effects      . Many hydrocarbons are very hydrophobic and their persistence within the ecosystem is due largely to their hydrophobicity and low water solubility  . Exposure of aquatic habitats and organisms to whole oil and toxic components causes acute mortality and chronic effects that can be felt during several years    .
Some bacteria found in contaminated environments with oil show the ability to metabolize many hydrocarbons and mineralizing this organic matter  . The degradation of many hydrocarbon compounds is known to be mediated by plasmid encoded enzymes  . The bacteria known to degrade PAHs and others hydrocarbons have many of the genes often located on plasmids  . A plasmid may encode a complete degradative pathway or partial degradative step. Some plasmids may allow the host to grow on several hydrocarbons as sole carbon and energy sources  .
The importance of HGT is strictly related with the construction of new plasmids and genomes to allow the evolutionary success  . Bosma et al. (2001)  showed that slightly differs from homologous operons, encoding for catabolic pathways have been found frequently in phylogenetically distant organisms, suggesting the occurrence of extensive HGT. This work aimed demonstrates the utility of molecular phylogenetic methods in studies of horizontal transfer of functional genes of crude oil degradative pathways in bacteria.
2. Materials and Methods
2.1. Nucleotide Sequences
The complete nucleotide sequences of 16S rRNA genes used in present study (Table 1) were obtained from European Molecular Biology Laboratory―EMBL (http://www.ebi.ac.uk/genomes/bacteria.html) and the others sequences were obtained from the National Center for Biotechnology Information-GenBank (http://www.ncbi.nlm.nih.gov). For analyses involving the alkB and catA genes, 12 sequences were selected (Table 1) from 12 different bacterial genus. Others two housekeeping genes were searched, rpoD and fabD genes (Table 2) for the same bacterial genus. Functionally, the 16S, housekeeping genes and catabolic genes are unrelated to the others. The choices were made to evaluate the phylogenetic relationships of these taxonomic groups and try to relate them.
2.2. Phylogenetic Analysis
Previously all the sequences, except of 16S rRNA, were translated into amino
Table 1. Bacterial genus and NCBI accession number of rpoD, fabD, alkB and catA sequences used in this work.
Table 2. Bacterial genus names and EMBL accession number of 16S rRNA sequences used.
acid sequences for to do the aligning with Clustal W in MEGA 6.0 software  . The individuals’ phylogenetic hypothesis were performed with Baysian inference  with MrBayes 3.1  to the five set of genes. To infer the tree through the Bayesian inference made two independent analyses with four chains each, one cold and three hot chains. Bayesian phylogenetic analyses were conducted by Markov Chain Monte Carlo simulations (MCMC) for 10 million generations. The likelihood was checked after burned-out 25% of the trees and to construct the consensus tree.
3. Results and Discussion
The Bayesian Inference hypotheses for the 16S gene are shown in Figure 1, which presents the expected pattern of genus.
In the Figure 2 are showed the Bayesian Inference hypotheses for the rpoD and fabD housekeeping genes. The topologies are different among both tree and these with 16S gene tree. Gordonia, Nocardia and Rhodococcus are very near genus, however, appear no so much near on the rpoD tree, these genus appear in over half of the disrupted mycobacterial clans, suggesting relatively short-distance transfers of genetic material either into or out mycobacterial species  . The genus Gordonia and Nocardia are distinct only by ability of Gordonia representants may reduce nitrate, which not occur with Nocardia   .
It is expected that molecular phylogenies based on single genes lead to apparently conflicting results with alternative branches  . This conflict is also observed in phylogenetic tree constructed from hydrocarbons catabolism genes, such as alkB and catA genes (Figure 3).
The conflicting topologies shown in Figures 1-3, with very different branches, suggesting that the all the four genes (rpoD, fabD, alkB, and catA) were indeed subjected to horizontal transfer events among these bacterial genus. Yamamoto
Figure 1. Tree obtained by Bayesian Inference analysis from sequences of the 16S gene.
Figure 2. Trees obtained by Bayesian Inference analysis from sequences of the rpoD (left) and fabD (right) housekeeping genes.
Figure 3. Trees obtained by Baysean Inference analysis from sequences of the alkB (left) and catA (right) hydrocarbons catabolism genes.
& Harayama (1998)  suggested that the genetic distances among two housekeeping genes may be caused by single-event mutations introducing multiple substitutions. Soler et al. (2004)  observed that exist distinct nucleotide substitution rates between gyrB and rpoD sequences, so it may be a significant factor to driving the evolution of two housekeeping genes. In addition the possibility of horizontal gene transfer involving housekeeping genes not very likely, it must be taken into account. Because these doubts, the study of several housekeeping gene sequences and multilocus analysis has already been recommended for improving the reliability of phylogenetic inference   . Soler et al. (2004)  proved that rpoD and gyrB housekeeping genes, independently, are excellent molecular makers for assessing phylogeny in the genus Aeromonas, which suggest that horizontal transfer maybe not occur between closely species, but yet may occur between other taxonomic levels.
Hydrocarbon catabolism genes are largely distributed in bacteria and many of them are located in plasmids    . Many studies showed HGT occurring between bacteria with genes involved with catabolism of distinct hydrocarbons and plasmids are closely related with this process    , this may explain the divergence of topologies of trees obtained by Bayesian Inference analysis from sequences of the hydrocarbon catabolism and housekeeping genes. Polycyclic aromatic hydrocarbon (PAH) degrading bacteria are known by its ability of transfer horizontally these genes inside the community, one example is the Catechol 1,2-Dioxygenase (catA)    .
Bacterial alkane degradation is important for the bioremediation of petroleum-contaminated environments since these compounds are predominant in crude oil   . A number of bacteria have multiple alkane hydroxylase genes which proven to potentially expand the n-alkane range of the host strain  . More than 60 genus of aerobic bacteria and 5 genus of anaerobic bacteria have been reported to be able to degrade n-alkanes   . Some Rhodococcus strains are known to contain more than one alkB homologous genes, which have different substrate ranges and induction styles   . These facts corroborate with the ability of the alkane hydroxylase genes to be spread in the microbial community by HGT.
High molecular weight hydrocarbons and the PAH caused chronic effects on the biota, because its recalcitrant properties, low solubility and hard to volatilization makes it will remain a long time on the environment   . The PAH may be adsorbed to particular matter in suspension on water and consequently deposited in the sediment, which become a contaminant reservoir, playing ecotoxicological effects to marine biota   . The HGT may be used with the purpose to spread hydrocarbon catabolism genes within of bacterial community to metabolyze faster the environmental contaminants in marine ecosystems.
The genotypic plasticity ability of the bacterial community to adapt on different environments is a great evidence of the occurrence of horizontal gene transfer in nature. On this way, we conclude that phylogenetic tools can be useful for inferring horizontal transfer events through the comparison with tree obtained by 16S rRNA sequence analysis. In this way, hydrocarbons catabolism genes as well housekeeping genes can be subject to horizontal gene transfers. The spreading of catabolic routes genes on the bacterial community provides a great diversity of ecological niches. In this way, bioaugmentation strategies can consider the ability of HGT to accelerate the hydrocarbons degradation in natural environments.
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