Author(s): Diogo Jurelevicius 1, Vanessa Marques Alvarez 1, Raquel Peixoto 2, Alexandre S. Rosado 2, Lucy Seldin 1,*
Alkane-utilizing bacteria are widespread in marine and terrestrial environments , . These bacteria generally possess the Alk enzyme system, which is involved in the metabolic pathway for the degradation of alkanes, the main compounds found in petroleum and its derivatives . The functional Alk enzyme system comprises the transmembrane alkane monooxygenase AlkB (encoded by the alkB gene and involved in the initial activation step of aerobic aliphatic hydrocarbon metabolism) and two co-factors named rubredoxin ( alkF ) and rubredoxin reductase (alkG ). These co-factors are responsible for transferring the electrons involved in alkane hydroxylation by AlkB . In other studies, the alkB gene has been used as a biomarker for the determination of the abundance and diversity of alkane-degrading bacteria [5-7].
Bacteria that possess the Alk enzyme system are valuable in environmental bioremediation and biocatalysis for the synthesis of industrial compounds, including drugs, pravastatin, and other compounds . The use of alkane-degrading bacteria in bioprocesses to produce valuable chemicals by transforming alkanes from hydrocarbon-contaminated samples is considered one of the most important biotechnological applications of these bacteria . Therefore, many studies report the detection and further characterization of alkB genes in a wide variety of bacterial genera , , .
However, the alkB genes characterized thus far may only represent a small fraction of the diversity found in natural environments and a limited biotechnological potential of alkane-degrading bacteria. Kuhn and co-workers  found new alkB genes when contaminated and uncontaminated marine sediments in Admiralty Bay, King George Island, Antarctica were analyzed. Similar results were obtained for samples from the Timor Sea in Australia  and from chronically polluted, sub Antarctic coastal sediments .
Although all AlkB proteins share considerable sequence homology, the nucleotide sequences encoding the alkB gene vary widely within the alkB -containing bacteria. To overcome this limitation and study the alkB diversity in natural environments, different alkB primers have been described , , . However, each set of primers is specific for primarily one group of bacteria [15-17], and designing broad-ranging alkB primers is not an easy task. Consequently, the presence and diversity of alkB sequences have likely been underestimated in the environmental samples previously studied.
In this study, we propose to use a combination of primers, rather than designing new primers, to improve the identification of the alkB diversity in different environments. For this purpose, we retrieved several pairs of alkB -targeting primers from the literature and tested their specificities against alkane-degrading bacteria previously isolated by our research group. These bacteria are representatives of Proteobacteria, Actinobacteria, Flavobacteria and Firmicutes groups. After selecting the combination of alkB -targeting primers showing the broadest coverage among the bacterial strains tested, we constructed alkB clone libraries using an oil-contaminated semiarid soil from Carmópolis, located in Sergipe (SE), Brazil to test the approach proposed here in the environment. Moreover, this strategy was also used to understand the alkB diversity in three pristine soil samples...