Metagenomics Reveals Diversity and Abundance of Meta-Cleavage
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Environmental Microbiology (2009) 11(9), 2216–2227 doi:10.1111/j.1462-2920.2009.01943.x Metagenomics reveals diversity and abundance of meta-cleavage pathways in microbial communities from soil highly contaminated with jet fuel under air-sparging bioremediation Maria V. Brennerova,1* Jirina Josefiova,1 supposed, attaining a complementary and community- Vladimir Brenner,1 Dietmar H. Pieper2 and balanced catalytic power against diverse catecholic Howard Junca2† derivatives, as necessary for effective degradation of 1Institute of Microbiology, Czech Academy of Sciences, mixtures of aromatics. Prague, Czech Republic. 2Department of Microbial Pathogenesis, HZI – Introduction Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany. Microbial communities are fundamental components of ecosystems, playing critical roles in the metabolism of organic matter. They are predominantly involved in detoxi- Summaryemi_1943 2216..2227 fication of contaminated sites and organisms degrading a The extradiol dioxygenase diversity of a site highly wide range of pollutants have been described. In nearly contaminated with aliphatic and aromatic hydrocar- all situations, microbial reactions are the dominant bons under air-sparging treatment was assessed by processes driving natural attenuation or bioremediation functional screening of a fosmid library in Escherichia processes. coli with catechol as substrate. The 235 positive clones Aromatic hydrocarbons such as benzene, toluene from inserts of DNA extracted from contaminated and ethylbenzene are common contaminants of soil soil were equivalent to one extradiol dioxygenase- and groundwater (Witzig et al., 2006) and are listed as encoding gene per 3.6 Mb of DNA screened, indicating priority pollutants by the US Environmental Protection a strong selection for genes encoding this function. Agency (http://www.epa.gov/waterscience/criteria/ Three subfamilies were identified as being predomi- wqctable). Many microorganisms have evolved special- nant, with 72, 55 and 43 fosmid inserts carrying genes, ized pathways to use aromatic compounds as their sole related to those encoding TbuE of Ralstonia pickettii carbon and energy sources (Harwood and Parales, 1996; PK01 (EXDO-D), IpbC of Pseudomonas sp. JR1 (EXDO- Pieper, 2005). Metabolic pathways and encoding genes K2) or DbtC of Burkholderia sp. DBT1 (EXDO-Dbt), have been characterized in many bacterial strains, pre- respectively, whereas genes encoding enzymes dominantly Proteobacteria and Actinobacteria. In the case related to XylE of Pseudomonas putida mt-2 were not of hydrophobic pollutants such as benzene, toluene, naph- observed. Genes encoding oxygenases related to iso- thalene, biphenyl or polycyclic aromatics, aerobic degra- propylbenzene dioxygenases were usually colocal- dation is usually initiated by activation of the aromatic ring ized with genes encoding EXDO-K2 dioxygenases. through oxygenation reactions catalysed by Rieske non- Functional analysis of representative proteins indi- haem iron oxygenases (Gibson and Parales, 2000) or, as cated a subcluster of EXDO-D proteins to show ex- intensively described for toluene degradation, through ceptional high affinity towards different catecholic members of the soluble diiron family of mono- substrates. Based on Vmax/Km specificity constants, a oxygenases (Leahy et al., 2003). task-sharing between different extradiol dioxygenases While a relatively broad diversity of activation mecha- in the community of the contaminated site can be nisms is possible in aromatic degradation, these path- ways usually converge in the formation of a few, usually Received 26 January, 2009; revised 19 March, 2009; accepted 24 dihydroxylated central intermediates, which are sub- March 2009. *For correspondence. E-mail [email protected]; Tel. (+420) 241062781; Fax (+420) 241722257. †Present address: strates for ring-cleavage dioxygenases. Methylsubstituted Molecular Genetics & Biotechnology – CorpoGen Corporation, aromatics such as toluene or xylenes and aromatics con- Bogotá, Colombia. taining two or more aromatic ring systems are usually Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/ degraded via the respective dihydroxylated derivatives authorresources/onlineopen.html and extradiol cleavage (Eltis and Bolin, 1996). Extradiol © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd Active meta-cleavage pathways 2217 dioxygenases are thus key enzymes in the degradation of nated area covers more than 28 ha with concentrations of aromatic compounds and many of such proteins and their total petroleum hydrocarbons ranging from 5000 to encoding sequences have been described, purified and 55 000 mg kg-1 of dry soil. Significant amounts of aro- characterized in the last decades. Examination of their matic hydrocarbons were detected and the concentration relationships has shown that the extradiol dioxygenases of BTEX (benzene, toluene, ethylbenzene, xylenes) was can be categorized into three types belonging to different reaching 1000 mg kg-1 of dry soil (Machackova et al., families. Type I enzymes belong to the vicinal oxygen 2008). Parts of the area have been under air-sparging chelate family (Eltis and Bolin, 1996), type II enzymes treatment for 5 years and thus, the in situ stimulation of are related to LigB protocatechuate 4,5-dioxygenase the natural microflora has presented us with an extraor- (Sugimoto et al., 1999) and type III enzymes belong to dinary opportunity to analyse the catabolic diversity for the cupin superfamily (Dunwell et al., 2000). Importantly, aromatic degradation that has been developed under novel extradiol dioxygenases are constantly being real-site conditions. reported (Nogales et al., 2005; Moonen et al., 2008). In this study we used soil from the Hradcany area as a Most of our knowledge on how pollutants are degraded source for metagenomic DNA to characterize the diversity in the environment has been obtained by laboratory of genes encoding catechol 2,3-dioxygenases by a studies using isolated pollutant-degrading microorganisms culture-independent function-based screening approach. or model communities. However, various culture- independent studies on genes coding for critical steps in aromatic degradation such as ring activation, or cleavage Results suggest that distinct and potentially numerically dominant Development and screening of metagenomic libraries sequence types of as-yet-unrecognized functional genes exist in natural and anthropogenically influenced environ- High-molecular-weight DNA was extracted from two soil ments and that the sequences from cultured strains are not samples representing both a highly polluted saturated likely to represent the microbial functional gene diversity in zone in close vicinity to the water table (S) and an unsat- the environment (Yeates et al., 2000; Witzig et al., 2006). urated zone with low level of contamination (W), which Most culture-independent surveys of catabolic gene diver- had bacterial densities culturable on R2A agar of 3.3 ¥ 106 sity in contaminated environments rely on conserved and 6.3 ¥ 105 colony-forming units (cfu) per gram of nucleotide sequences used for the design of primers to dry soil. The metagenomic libraries were generated survey the presence, abundance and diversity of catabolic in Escherichia coli using pCCFOS fosmid vector and genes encoding a defined group of enzymes supposed to contained 8.7 ¥ 104 and 3 ¥ 105 clones respectively. be critical in the target environment. However, such defini- Both libraries were screened for catechol 2,3- tion of critical enzymes is often based on knowledge using dioxygenase activity by spraying with catechol. In the isolates. As an example from culture-dependent studies, absence of inducer, yellow coloration due to the formation oxygenases of the benzene/toluene subfamily of Rieske of 2-hydroxymuconic semialdehyde was not observed. non-haem iron oxygenases would be expected to be However, 235 of 2.6 ¥ 104 clones from library S turned important in sites contaminated with benzene/toluene, yellow on plates supplemented with L-arabinose, indicat- whereas culture-independent studies covering a broad ing that the high-copy amplification feature of the copy- range of Rieske non-haem iron oxygenases discovered controlled fosmid vector facilitated the visual detection of isopropylbenzene dioxygenases to be abundant in a C23O expression. Whereas 0.91% of library S fosmid- respective contaminated site (Witzig et al., 2006). containing E. coli strains exhibited C23O activity, only 19 One approach that relies neither on conserved nucle- of 3.5 ¥ 104 library W fosmid-containing E. coli strains otide sequences nor on previous knowledge of isolates is (0.05%) were capable of cleaving catechol. This indicated the use of genomic libraries to retrieve genes catalysing a C23O gene abundance to be correlated with the contami- desired function from natural bacterial communities nation level and a selective advantage of bacteria har- without cultivation. In fact, functional screening of metage- bouring such function under the given environmental nomic libraries has led to the discovery of novel genes conditions. encoding antibiotic resistance (Riesenfeld et al., 2004), Diversity and average insert size of the generated ester- and glycosyl-hydrolases (Ferrer et al., 2005), libraries were characterized by PFGE of NotI-digested polyphenol oxidase (Beloqui et al., 2006) and also given plasmid DNA isolated from