To Be Submitted to Applied and Environmental Microbiology

1 1 Submitted to Microbiology 2 3 GENUINE GENETIC REDUNDANCY IN MALEYLACETATE REDUCTASE ENCODING 4 GENES INVOLVED IN DEGRADATION OF HALOAROMATIC COMPOUNDS BY 5 CUPRIAVIDUS NECATOR JMP134. 6 7 Running title: Maleylacetate reductases in Cupriavidus necator JMP134 8 Contents category: Physiology and Biochemistry. 9 10 Danilo Pérez-Pantoja, Raúl Donoso, Miguel A. Sánchez and Bernardo González#,* 11 12Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, P. 13Universidad Católica de Chile. Millennium Nucleus on Microbial Ecology and Environmental 14Microbiology and Biotechnology. NM-EMBA. Center for Advanced Studies in Ecology and 15Biodiversity. CASEB. Santiago, CHILE. Facultad# de Ingeniería y Ciencia, Universidad Adolfo 16Ibáñez. Santiago, CHILE. 17 18Keywords: Maleylacetate reductase, Cupriavidus necator JMP134, genetic redundancy, 19haloaromatic compounds, 2,4-dichlorophenoxyacetic acid. 20Abbreviations: 2-CMA: 2-chloromaleylacetate; 2,4-D: 2,4-dichlorophenoxyacetate; 2,4,6-TCP: 212,4,6-trichlorophenol; 3-CB: 3-chlorobenzoate; 4-FB: 4-fluorobenzoate; LB: Luria-Bertani; MA: 22maleylacetate; MAR: maleylacetate reductases 23 24*Corresponding author. Mailing address: Facultad de Ingeniería y Ciencia. Universidad Adolfo 25Ibáñez. Postal Code: 7941169. Santiago, Chile. Phone: 56-2-3311619. [email protected]. 2 1 SUMMARY 2 Maleylacetate reductases (MAR) are required for biodegradation of several 3substituted aromatic compounds. So far, the functionality of two MAR-encoding genes 4(tfdFI and tfdFII) has been reported in Cupriavidus necator JMP134 (pJP4), a known 5degrader of aromatic compounds. These two genes are encoded in the tfd gene 6clusters involved in turnover of 2,4-dichlorophenoxyacetate (2,4-D), and 3- 7chlorobenzoate (3-CB). The C. necator JMP134 genome comprises at least three other 8genes putatively encoding MAR (tcpD, hqoD and hxqD), but confirmation of their 9functionality and their role in the catabolism of haloaromatic compounds has not been 10assessed. RT-PCR expression analyses of C. necator JMP134 cells exposed to 2,4-D, 113-CB, 2,4,6-trichlorophenol (2,4,6-TCP) or 4-fluorobenzoate (4-FB) showed that tfdFI 12and tfdFII are induced by haloaromatics channeled to halocatechols as intermediates. In 13contrast, 2,4,6-TCP only induces tcpD, and any haloaromatic compounds tested did not 14induce hxqD and hqoD. However, the TcpD, HxqD and HqoD gene products showed 15MAR activity in cells extracts and provided the MAR function for 2,4-D catabolism when 16heterologously expressed in MAR-lacking strains. Growth tests for mutants of the five 17MAR-encoding genes in strain JMP134 showed that none of these genes is essential 18for degradation of the tested compounds. However, the role of tfdFI/tfdFII and tcpD 19genes in the expression of MAR activity during catabolism of 2,4-D and 2,4,6-TCP, 20respectively, was confirmed by enzyme activity tests in mutants. These results reveal a 21striking example of genetic redundancy in the degradation of aromatic compounds. 1 2 1 INTRODUCTION 2 3 Maleylacetate reductases (MAR) play an important role in degradation of 4(halo)aromatic compounds in fungi (Gaal & Neujar, 1980; Jones et al., 1995; Patel et 5al., 1992; Sparnins et al., 1979), Actinobacteria (Huang et al., 2006; Perry & Zylstra, 62007; Seibert et al., 1998; Travkin et al., 1999) and Proteobacteria (Armengaud et al., 71999; Daubaras et al., 1996; Endo et al., 2005; Moonen et al., 2008; Nikodem et al., 82003; Seibert et al., 2004; Yoshida et al., 2007). In these microorganisms, 9maleylacetate (MA) or 2-chloromaleylacetate (2-CMA) are generated during turnover of 10halocatechols, hydroxyquinol, chlorohydroxyquinol, hydroquinone and 11chlorohydroquinone, as ring-cleavage intermediates of a broad array of metabolized 12compounds. 13 MA is transformed to 3-oxoadipate through a NAD(P)H-dependent reduction of 14the carbon-carbon double bond catalyzed by MAR (Fig. 1A). In the case of 2-CMA, 15MAR initially catalyzes the NAD(P)H-dependent dechlorination to MA, which is then 16reduced to 3-oxoadipate (Fig. 1A). Therefore, two moles of NAD(P)H per mole of 17substrate are consumed during the MAR-mediated conversion of 2-CMA to 3- 18oxoadipate, which is subsequently funneled to the Krebs’s cycle (Kaschabek & 19Reineke, 1992; Kaschabeck & Reineke, 1995; Vollmer et al., 1993). 20 Cupriavidus necator JMP134 is a β -proteobacterium able to grow on a wide 21range of aromatic and haloaromatic compounds as sole carbon and energy source 22(Pérez-Pantoja et al., 2008). The haloaromatic compounds catabolized by C. necator 23JMP134 include 2,4-dichlorophenoxyacetate (2,4-D), 3-chlorobenzoate (3-CB), 2,4,6- 1 3 1trichlorophenol (2,4,6-TCP) and 4-fluorobenzoate (4-FB) that through diverse catabolic 2steps, are converted to MA or 2-CMA (Fig. 1A) (Pérez-Pantoja et al., 2008). 3 The role of MAR in C. necator JMP134 has been well established in the turnover 4of 3,5-dichlorocatechol, 3- (and 4-) chlorocatechol, 4-fluorocatechol and 6- 5chlorohydroxyquinol, generated during mineralization of 2,4-D (Seibert et al., 1993; 6Vollmer et al., 1993), 3-CB (Laemmli et al., 2000; Pérez-Pantoja et al., 2000), 4-FB 7(Schlomann et al., 1990) and 2,4,6-TCP (Padilla et al., 2000; Sánchez & González, 82007), respectively. Two MAR-encoding genes tfdFI and tfdFII have been 9previously identified in C. necator JMP134, both belonging to specialized gene clusters 10for chlorocatechol turnover located on the pJP4 catabolic plasmid (Fig. 1B) (Trefault et 11al., 2004). The functionality of both genes and their expression during mineralization of 122,4-D and 3-CB has been confirmed (Kasberg et al., 1995; Laemmli et al., 2004; 13Laemmli et al. 2000; Pérez-Pantoja et al., 2000; Plumeier et al., 2002). Additionally, the 14presence of at least one chromosomally encoded MAR has been shown (Padilla et al., 152000; Pérez-Pantoja et al., 2000; Plumeier et al., 2002; Vollmer et al., 1993). Recently, 16the analysis of the C. necator JMP134 genome sequence has revealed the presence of 17three hypothetical MAR-encoding genes located in chromosomal replicons (Fig. 1B). 18These additional gene sequences have been termed tcpD (Sánchez & González, 192007), hqoD and hxqD, since they are clustered with genes putatively encoding 2,4,6- 20TCP, hydroquinone and hydroxyquinol-metabolizing oxygenases, respectively (Pérez- 21Pantoja et al., 2008); however, their possible function requires experimental 22confirmation. 1 4 1 In this work, the functionality of the chromosomal MAR-encoding genes of C. 2necator JMP134 was assessed. The results reveal a genuine genetic redundancy for 3MAR functions in the catabolism of haloaromatic compounds. 1 5 1 METHODS 2 3Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids 4used in this study are listed in Table I. C. necator JMP134 (pJP4) and its derivatives 5were grown at 30°C in mineral salts medium using 50 mM phosphate buffer (pH 7.5) 6(Dorn et al., 1974). The medium was supplemented with different haloaromatic 7compounds 2,4-D (2.5 mM), 3-CB (2.5 mM), 4-FB (2.5 mM) and 2,4,6-TCP (0.5 8mM) or fructose (10 – 20 mM) as sole carbon source, plus the appropriate 9antibiotics: kanamycin (100 µ g/mL) and/or gentamycin (20 µ g/mL) (Table I). B. 10phytofirmans PsJN (pJP4) and its derivatives were grown under the same conditions 11but with 2,4-D (2.5 mM), as growth compound plus gentamycin (20 µ g/mL) and L- 12arabinose (0.25 mM), as inducer of PBAD promoter in pBS1 derivatives (Table I). P. 13putida KT2442 and its derivatives were grown under the same conditions but with 14succinate (30 mM) plus gentamycin (20 µ g/mL) and L-arabinose (5 mM) (Table I). E. 15coli Mach 1™ (Invitrogen Life Technologies, Carlsbad, CA) was used routinely as 16plasmid host and was grown at 37ºC in Luria-Bertani (LB) medium plus the appropriate 17antibiotic: ampicillin (100 µ g/mL), kanamycin (100 µ g/mL) or gentamycin (20 18µ g/mL). Growth was determined by measuring the optical density at 600 nm (OD600). 19At least two replicates were performed for each growth measurement. 20 21Detection of transcripts by RT-PCR. Cells of C. necator JMP134 were grown 22overnight in minimal medium with 10 mM of fructose as carbon source. This culture 23was used to inoculate fresh medium and grown until OD600=0.7, subsequently 1 6 1supplemented with 0.25 mM of 3-CB, 2,4-D, 4-FB or 2,4,6-TCP and incubated for 1 h. 2Then, total RNA was obtained from 4 mL of the culture, using RNA protect bacteria 3reagent and RNeasy Mini Kit (QIAGEN Inc., Chatsworth, CA), according to 4manufacturer’s instructions. The obtained RNA was quantified using a GeneQuant™ 51300 Spectrophotometer (GE Healthcare, Piscataway, NJ) and treated with TURBO 6DNase Kit (Ambion, Austin, TX) to remove any DNA contamination, following the 7manufacturer’s instructions. The RT-PCR was carried out using ImProm-II Reverse 8Transcription System (Promega Corporation, Madison, WI) with 1 μg of total RNA in 20 9μL reactions. After reverse transcription, PCR amplifications were carried out using the 10following primer pairs: tfdFIintFW-tfdFIintRV, tfdFIIintFW-tfdFIIintRV, tcpDintFW2- 11tcpDintRV2, hqoDintFW-hqoDintRV and hxqDintFW-hxqDintRV (these and all primer 12pairs used in this work are shown in Table II); in a 25 μL mixture which contained 1 μL 13of total cDNA, 50 pmol of each primer, 50 μM of each deoxynucleoside triphosphate, 1 14mM MgCl2, and 5 U of Taq DNA polymerase, prepared in the reaction buffer supplied by 15the manufacturer. The temperature program was as follows: initial denaturation at 95°C 16for 5 min, then 28 cycles of 30 s at 95°C, 30 s at 60°C, and 60 s at 72°C, with a final 17extension at 72°C for 10 min. Negative control reactions were performed in the same 18way, except that reverse transcriptase was omitted in the reaction mixtures. 19 20Construction of plasmid derivatives expressing MAR-encoding genes.

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