Molecular, phylogenetic and functional characterization of Burkholderia sp. DBT1, a bacterial strain involved in polycyclic aromatic hydrocarbons (PAHs) degradation

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds that have accumulated in the natural environment mainly as a result of anthropogenic activities. These compounds are largely suspected to act as potential mutagens, teratogens as well as carcinogens. Moreover, their low aqueous solubility and, consequently, their low bioavailability are a great obstacle to microbial degradation. The word “bioremediation” has been used to describe the process of using microorganisms to degrade or remove hazardous compounds and wastes such as PAHs from the environment. Nevertheless, before developing an efficient protocol for bioremediation is necessary to perform an upstream study, in order to isolate, identify and characterize organisms suitable to this purpose. Burkholderia sp. DBT1 is a bacterial strain identified in an oil refinery wastewater which can degrade DBT nearly completely through the Kodama pathway within three days. Aimed to clarify the effective potential and actual utilization of such strain in bioremediation protocol, different aspects of B. sp. DBT1 have been investigated. - Identification of DNA sequences flanking p51 operon: Molecular characterization of strain DBT1 has showed that six of eight genes required for DBT degradation by Kodama pathway are present in two operons – p51 and pH1A. p51 genetic fragment harbor dbtC,Ab,ORF6,ORF7 replicative unit. The sequence of p51 genetic fragment stopped at the end of ORF 7 and the transcription terminator was not yet detected. Therefore, identification of DNA sequence flanking p51 operon was carried out in order to detect possible gene sequence downstream ORF 7. - Taxonomic analysis: Taxonomic status of Burkholderia sp. DBT1 has never been resolved. It is important to remark that several species of Burkholderia can cause food poisoning or can behave as plant and animal pathogens. Furthermore, different strains belonging to the Burkholderia cepacia complex (Bcc) are involved in life-threatening infections of humans such as cystic fibrosis. - Study of PAHs degrading activity: Since normally several organic pollutants contribute together to the contamination at different sites, isolation and characterization of microbes able to use a wide range of PAHs compounds as sole source of carbon and energy are of great interest in order to select useful candidates for applications in bioremediation. - Toxicology and pathogenic analysis: Several Burkholderia species are common soil inhabitants, nevertheless some species of this genus are known as toxin producer. Therefore, some tests will be carried out to even investigate the eco-toxicological safety of strain DBT1 for environmental use. - Study of endophytes bacterial strains: Once it is been clarified the metabolic, taxonomic aspect of strain DBT1, it results very important to set up an efficient protocol for a future application in open field (in situ). A new promise strategy is to use endophitic bacterial equipped with appropriate degradation pathway in order to improve in planta PAHs degradation. Thus, selection and study of bacterial strains able both to degrade PAHs and to colonize host plants (e.g. Aspen) in order to verify the bioremediation potential of plant-endophytic bacteria systems, using both molecular and physiologic analysis. The work here presented has demonstrated that the hydratase-aldolase and ferredoxin reductase sequences, the two genes missing, are collocated downstream the putative sequence ORF 7 within p51 operon. Thus, the missing enzymes have been recovered, demonstrating that the whole set of enzyme involved in Kodama pathway degradation is harbored in p51 and pH1A operons. Moreover, the results so far achieved suggest that the strain DBT1 presents a versatile metabolism towards PAHs, and this is an important trait for the possible use of this strain in environmental clean up. This strain is able to use fluorene, naphtalene, DBT and phenanthrene for growing, although growth on the first two compounds needs a pre-induction. Therefore, it can be suggested that phenanthrene is metabolized through 2-carboxycinnamic acid, phthalic acid and further metabilized in protocatechol. The PAHs degradation can occur even by a co-metabolism process. The application of bacterial strains in environmental clean up is severely subject to another crucial pre-requisites: the ecotoxicological safety of the selected strain and its probative exclusion from pathogenic species such as Burkholderia cepacia complex. The data presented in this study indicate that strain DBT1 can be considered a member of the species fungorum, for which the name Burkholderia fungorum strain DBT1 is here proposed. Since the original report, B. fungorum was identified in a wide range of environment such as soil, plant-associated samples, in infections of the central nervous system of a pig and a deer, and in the respiratory secretions of people with cystic fibrosis. Therefore, the investigation about possible toxic effects by strain DBT1 has been considered of prominent significance in the present study; and bioassay to detect mitochondrial toxicity in mammalian cells by JC-1 and PI staining of human cell lines would be useful to investigate toxic microbial metabolites. Results so far achieved show that B. DBT1 and B. fungorum type strain was unable to damage mitochondrial and human cells membranes. However, it is important to underline that this strategy does not rule out the possibility that the strain investigated might be pathogenic under specific circumstances or on different targets not yet ascertained, such as to substances affecting the synthesis of proteins or nucleic acids or their regulation. Besides, as cited before, the second strategy for a better use the bioremediation potential of plant-endophytic bacteria systems is focused on isolating, identifying and characterizing the bacteria that already exist as bacterial endophytes in plants and which are applyable to phytoremediation protocols. Poplar trees (Populus spp.) are commonly used as phytoremediation tools because they are perennial, hardy, tolerant to high concentration of organic compounds, highly tolerant to flooding, fast growing, easily propagated and have a wide range adaptation. Thus, a careful screening and characterization of endophytes from poplar plants growth on PAHs contaminated soils were carried out. The results obtained confirm that natural bacteria degrading recalcitrant compounds are largely present among endophytic populations of plants growth in contaminated sites, this could mean that endophytes have a role in metabolizing these substances. Moreover, Burkholderia is resulted be the genera mainly involved in PAHs degradation, and fungorum the species more representative. Interestingly, the majorities of these bacteria are involved in phenanthrene and DBT degradation, and harbor p51 and pH1A, the operons formerly identify in DBT1 strain. However, the subsequent DGGE and restriction analysis on these DNA fragments, showed the presence of a single genotype among the endophytic bacteria, but different from DBT1. The difference between strains isolated in Finland and DBT1 is not only at genetic level. In fact, B. sp. DBT1 resulted to be more efficient in the degradation towards PAHs tested, especially in the metabolism of DBT. Moreover, the results suggest the application of B. sp. DBT1 as poplar endophyte. Eventually, the results obtained from this thesis work incentives the use of the strain Burkholderia fungorum DBT1 for a possible exploitation in bioremediation protocols of PHA-contaminated sites; moreover it suggest an actual use of such strain as endophytes in poplar plant in order to improve in planta degradation.