ThnA3 negatively modulates thn operon expression

In order to characterize the elements involved in regulation of thn genes expression further, we have reconstructed the regulation and the modulation mediated by ThnA3 in a more defined genetic background by using the mutant strain T690-690. Strain T690 has a 12.2-kb deletion covering from thnG to thnY (see Figure 1), which have been replaced by a kanamycin (Km) resistance cassette [10]. As a consequence this strain is unable to grow with tetralin as the sole source of carbon and energy. Besides the 12.2-kb deletion, T690-690 also bears a thnC::lacZ translational fusion integrated in its chromosome, which allows measurement of thn gene expression [5,10].

Previous experiments had shown that expression of the thnA1A2A3A4 genes in T690 allowed the initial dioxygenation reaction of the benzene ring of tetralin, since the dioxygenase product, 1,2-dihydroxy-1,2,5,6,7,8-hexahydronaphthalene was produced upon addition of tetralin [10]. We have complemented T690-690 with the isopropyl β-D-thiogalactopyranoside-inducible expression vector pIZ1016 in which we sequentially cloned each of the regulatory elements, thnR and thnY, in addition to the components of the initial dioxygenase complex, encoding either only the electron transport proteins ThnA3 and ThnA4, or the complete dioxygenase complex ThnA1, ThnA2, ThnA3 and ThnA4.

To analyse the effect of these proteins on thn expression, plasmids pIZ1017 (thnR), pMPO750 (thnY), pIZ1158 (thnRY), pMPO751 (thnA3A4RY) and pMPO754 (thnA1A2A3A4RY), were introduced into T690-690 and the levels of β-galactosidase activity from the thnC::lacZ translational fusion were measured after growth under carbon-limiting conditions with 8 mM of β-hydroxybutyrate (βHB) and in the presence of either tetralin or trans-decalin as examples of efficient and inefficient inducers, respectively (Figure 2). The wild type strain, with and without pIZ1016, mutants of TFA lacking the ferredoxin ThnA3 and the α subunit of the dioxygenase (ThnA1) (left part of the figure), and T690-690 under the same inducing conditions were included as controls.

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Figure 2. Expression of a thnC-lacZ fusion in the T690-690 mutant strain carrying different combinations of the thnRY regulatory genes plus the components of the initial dioxygenase thnA3A4 and thnA1A2A3A4 from P_tac.

β-galactosidase activity was assayed after growth on 8 mM β-hydroxybutyrate (carbon-limiting conditions), in the absence of inducers (white bars), in the presence of tetralin (black bars), or trans-decalin (grey bars). Expression levels of the wild type strain without and with the empty vector pIZ1016 are shown. The mutants of TFA lacking the ferredoxin ThnA3 and the α subunit of the dioxygenase (ThnA1) are also included.

https://doi.org/10.1371/journal.pone.0073910.g002

As expected, neither the T690-690 mutant nor its derivative strains expressing thnR or thnY alone were able to activate expression of thnC::lacZ, in agreement with previous reports indicating that both proteins are strictly required for expression of tetralin biodegradation genes [1]. Co-expression of both thnR and thnY from the P_tac promoter produced very high levels of β-galactosidase activity no matter what molecule was used to induce the system, indicating that under these circumstances the regulatory system, though responsive to the effector molecules, had completely lost the ability to discriminate between them. This is essentially the same expression phenotype observed for mutant strains lacking the ferredoxin ThnA3 [3]

To analyse the effect of adding ThnA3 on thn gene expression, T690-690 was complemented with plasmid pMPO751, which carries genes encoding ThnA3 and the ferredoxin reductase ThnA4, its electron donor partner, in addition to the regulatory proteins ThnR and ThnY. As shown in Figure 2, induction of thn gene expression by either tetralin or trans-decalin was clearly reduced. These results are in complete agreement with the expression phenotype of thnA1 mutants, which lack dioxygenase activity, in which presumably an intracellular accumulation of reduced ThnA3 maintains thn gene expression at a very low level even in the presence of tetralin [3]. When all the components required for initial dioxygenation of tetralin (thnA1A2A3A4) were included, in addition to the regulatory genes, the wild type expression profile, i.e. high induction levels using tetralin and low levels using trans-decalin, was completely restored (Figure 2).

Taken together, these results identify the elements that influence the levels of thn gene expression and clearly indicate that the ferredoxin ThnA3 has a discriminatory role among potential inducers, preventing transcription under conditions that presumably lead to accumulation of the reduced form of the protein. This situation may be found in the wild type strain in the absence of a suitable substrate for the dioxygenase (complementation with pMPO754 with trans-decalin), or in a mutant lacking tetralin dioxygenase (complementation with pMPO751 even with tetralin). In summary, this finding confirms the discriminatory function of ThnA3 and the negative role of its reduced form.

Construction of thnY mutants altered in electron transport cofactors

As previously reported, ThnY does not contain all the properties of its ferredoxin reductase homologs, but it possesses functional [2Fe-2S] cluster and FAD prosthetic groups, as they are able to undergo reduction in the presence of sodium dithionite [4]. Thus, one possibility is that negative modulation by the reduced form of ThnA3 is exerted through redox changes in ThnY, which in turn, would affect its function in activating thn gene expression.

In order to investigate whether the electron transfer properties of ThnY are important for its function as a regulatory protein, and to establish a relation between its structure and function, codons encoding conserved amino acid residues critical for binding of the prosthetic groups were mutated by site-directed mutagenesis, as detailed in Materials and Methods. Residues Cys³⁵ and Cys⁴⁰ of ThnY, corresponding to the highly conserved residues C-X4-C-X2-C-X29/30-C for plant-type [2Fe-2S] cluster binding, were substituted by serine, a structural analog of cysteine. Amino acids Arg¹³⁷ and Tyr¹³⁹ of ThnY, corresponding to the highly conserved residues RXYS are of particular importance for binding of the isoalloxazine ring of the flavin [11], and therefore these residues were exchanged for Leu, an amino acid with an aliphatic side chain. These changes should disrupt the hydrogen-bonding network to the FAD. Although the NAD(P) H-binding domain of ThnY is still recognizable, amino acid sequence alignment at the position of the glycine signature (GGXGXXP), proposed to be involved in the binding of the adenosine-5´-phosphate groups of NADH or 2´-phospho-AMP of NADPH [12], showed that the first two glycines are occupied by asparagines (N200,N201) and the conserved proline is replaced by a serine (S206) [4]. Given that a deletion of the whole NAD(P) H domain resulted in non-functional ThnY (not shown), suggesting that it might have a role controlling the regulatory function of ThnY, the corresponding N201 and S206 residues of ThnY were changed to the highly conserved glycine and proline, respectively. Figure 3 shows a diagrammatic representation of the domain structure and consensus peptide segments involved in the plant-type [2Fe-2S] cluster, flavin and NAD(P) H-binding motifs in representative ferredoxin reductases of dioxygenases systems [9], where the locations of the mutations constructed in this study are also indicated.

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Figure 3. Diagrammatic representation of the three-domain structure of ferredoxin reductase representatives of Classes IB and III of dioxygenases systems.

BenC, benzoate 1,2-dioxygenase reductase from Acinetobacter sp. [11] and NdoR, naphthalene dioxygenase reductase from Pseudomonas [25]. Conserved sequences for the three cofactors binding domains of those reductases and the identified conserved motifs in ThnY are shown. The conserved cysteines for plant-type [2Fe-2S] cluster are shaded and in bold; conserved residues for binding of isoalloxazine and phosphate groups of the FAD, respectively, are shown in bold. The highly conserved glycine fingerprints involved in binding of NAD(P) H in plant/bacterial-type FNRs (ferredoxin:NAD(P)⁺ reductases) are shaded; other conserved amino acid and the aromatic residue involved in the interaction with the adenine moiety of FAD are also indicated in bold [4]. Amino acid replacements characterized in this study are indicated by arrows.

https://doi.org/10.1371/journal.pone.0073910.g003

Effect of thnY mutations on the regulation of thn expression

To examine the in vivo effect of the ThnY amino acid variants on transcriptional activation by ThnR, the T601-1002 strain, which carries a mini-Tn5km inserted into codon 140 of thnY and a thnC::lacZ translational fusion [1] also inserted into the chromosome, was complemented with the pIZ1016 derivative plasmids pMPO750, expressing wild type thnY and pMPO764, pMPO765, pMPO757, pMPO756 and pMPO763, expressing the mutant derivatives C35S, C40S, R137L, Y139L and N201G,S206P, respectively. These constructs were designed to place the expression of thnY alleles under the control of the P_tac promoter. All plasmids resulting from the inducible expression vector pIZ1016 tend to integrate into the TFA chromosome, introducing the mutated DNA regions into its chromosome (data not shown). Therefore, those colonies that had placed the thnY alleles under the control of the P_tac promoter after the first recombination event were selected by PCR. A schematic representation of the final genome organization around the thnR and thnY region is shown in Figure 4A. The effect of these ThnY variants on thn gene expression, as well as their capacity of distinguishing between a good and a bad substrate of the catabolic pathway, was determined under carbon-limiting conditions using either tetralin or trans-decalin as inducers and with or without IPTG, as indicated in Figure 4A. The levels of the respective ThnY protein variants in each strain and condition are also shown in Figure 4B. T601-1002 was unable to grow with tetralin as the sole source of carbon and energy and to induce expression of the thnC-lacZ fusion (data not shown). Plasmid pMPO750, expressing WT ThnY, complemented the thnY mutation because it allowed the mutant strain to grow on tetralin as the only carbon and energy source (not shown), and restored the levels and expression profile to those of the wild type TFA strain (Figure 4A). The thnC-lacZ expression levels were similar regardless of whether IPTG was added, even though the concentration of ThnY in the absence of IPTG was very low, much lower than that produced by the wild type TFA strain (Figure 4B). Thus, it appears that the ThnY concentration even in the absence of IPTG was sufficient to obtain maximal induction levels (Figure 4).

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Figure 4. Effect of ThnY variants on thn gene expression.

(A) Cultures were grown under carbon-limiting conditions using the following conditions: tetralin (black bars); trans-decalin (grey bars); tetralin plus IPTG (black hatched bars); and trans-decalin plus IPTG (grey cross-hatched bars). β-galactosidase activity was measured as described in Materials and Methods. Values represent the means of at least three independent determinations. The following plasmids expressing either wild type or ThnY variants were used for complementation of mutant strain T601-1002: pMPO750 (wild-type ThnY), pMPO764 (ThnY-C35S), pMPO765 (ThnY-C40S), pMPO757 (ThnY-R137L), pMPO756 (ThnY-Y139L), pMPO763 (ThnY-N201G,S206P). (+) and (-) indicate addition or not of IPTG. A schematic representation of the thnRY region after the recombination event leading to the integration of the thnY derivatives into the chromosome is represented on the top. thnY** denotes WT thnY or its mutant derivatives. (B) Western blot analysis showing the level of ThnY accumulated in the tetralin-induced cultures used for the β-galactosidase assays with (+) and without (-) IPTG. This allows direct comparison of the accumulated levels of ThnY since the amount of total protein in each lane is the same. ThnY synthesis in the wild type TFA, (1), and in T601–1002, (2), was included for comparison.

https://doi.org/10.1371/journal.pone.0073910.g004

The absolute induction levels using ThnY-R137L were clearly lower than those using WT ThnY. This low level is consistent with the slower growth of the strain and reduced total biomass in the presence of tetralin (not shown), as compared to the WT ThnY. Western blots showed that production of ThnY-R137L was approximately similar to that of the WT ThnY (Figure 4B), therefore, the lower expression levels using ThnY-R137L indicate that this protein was partially active. These results suggest that the R137 of ThnY plays an important role in thn transcriptional activation. However, the ThnY-R137L protein variant produced an induction profile similar to that of the WT ThnY, i.e. higher induction by tetralin than that by trans-decalin, thus indicating a discrimination capacity similar to that of WT ThnY.

Mutants ThnY-C35S and ThnY-Y139L performed as the wild type protein, since both were able to restore growth on tetralin (not shown) and gave similar levels of thn gene expression (Figure 4A). This indicates that their functionality was not substantially affected by the amino acid exchanges. However, they appear to be slightly affected in discrimination capacity between strong and weak inducers. For example, induction by trans-decalin was always higher than 50% of the induction by tetralin, as compared to the 25% that was obtained using WT ThnY. On the other hand, although the mutation thnY-N201G,S206P also did not affect the functionality of ThnY, the protein variant had clearly lost its discrimination capacity, since the expression levels induced by trans-decalin were very similar to those caused by tetralin (88% without and 106% with IPTG). As shown by the Western blots (Figure 4B) and the expression levels (Figure 4A) increasing the amount of ThnY-Y139L or ThnY-N201G,S206P by addition of IPTG did not result in a substantial increase in the levels of thn gene expression in the presence of tetralin, suggesting that, as in the case of the WT ThnY, the low concentration of these proteins even in the absence of IPTG was also saturating.

The ThnY-C40S variant had also lost its discrimination capacity between tetralin and trans-decalin either in the absence (induction by trans-decalin was 76% of that by tetralin) or in the presence of IPTG (induction by trans-decalin was 104% of that by tetralin) (Figure 4A). In this case, augmenting the amounts of ThnY-C40S by adding IPTG increased induction by tetralin to completely restore the wild type expression levels, thus indicating that ThnY function was not saturating in the absence of IPTG. Since accumulation of this protein variant was significantly lower than the WT ThnY (Figure 4B), we interpret this to mean that the lower induction of gene expression in the absence of IPTG does not indicate a limited functionality of ThnY-C40S but is rather due to a very low level of ThnY-C40S, which is undetectable under this condition.

Altogether the results presented here indicated that alterations in the redox cofactor binding domains of ThnY are not important for the activation function but rather are critical for the discriminatory function of the regulatory system. This effect is strongest in the case of the C40S and the double N201G,S206P amino acid substitutions since they have completely lost the ability to discriminate between tetralin and trans-decalin therefore activating the catabolic pathway under inappropriate conditions.

In order to confirm whether these two ThnY variants had altered the capacity to discriminate between different inducer molecules, we extended the analysis by examining induction by other molecules containing aromatic or alicyclic rings (Figure 5). As mentioned above for trans-decalin, complementation of the chromosomal thnY mutant strain (T601–1002) with WT thnY clearly showed that these molecules were obviously less efficient inducers than tetralin (40% for cis-decalin and cyclohexane, and 15% for benzene, numbers 2, 3 and 5 respectively in Figure 5). These data agree with those already observed with the wild type TFA [3]. In contrast, in the mutant strains expressing thnY-C40S and thnY-N201G,S206P, molecules such as cis-decalin and cyclohexane induced the tetralin biodegradation genes to levels substantially higher than those obtained with the WT thnY, and more similar to those obtained with tetralin, the true substrate of the pathway, (68-65% for the mutant ThnY-C40S and 81%-92% for the double mutant, respectively).

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Figure 5. Inducer profile for thn operon expression in ThnY-C40S and ThnY N201G,S206P variants.

The relative efficiency of different molecules to induce expression was measured by quantifying the levels of expression of a thnC::lacZ fusion under carbon limiting conditions containing IPTG and the indicated molecules as inducers. For each protein variant, induction obtained with tetralin was set to 100%. The ΔthnA3 strain under the same growth conditions is also included for comparison.

https://doi.org/10.1371/journal.pone.0073910.g005

As shown in Figure 5, this expression phenotype resembles that of mutant strains lacking the ferredoxin ThnA3, because they are able to activate the catabolic pathway to high levels in response to compounds other than tetralin. Overall, these results confirm that the C40S and N201G,S206P substitutions diminish the efficiency of the discriminatory response of the thn system.

Effect of the ferredoxin ThnA3 on the activity of ThnY-C40S and ThnY-N201G,S206P

It is clear from the results shown above that the variants ThnY-C40S and ThnY N201G,S206P cannot distinguish between different molecules, suggesting that they might escape the negative modulation exerted by the ferredoxin ThnA3. Therefore, we decided to examine the activities of these mutants in a strain lacking ThnA3 and in a strain presumably accumulating the ferredoxin ThnA3 in its reduced state, as is the case with the thnA1 point mutant, thnA1-D221A. The amino acid substitution Asp221Ala renders the tetralin dioxygenase fully inactive by apparently preventing electron transfer from the ferredoxin to the catalytic center and, consequently, to the substrate [13,14].

To that end, the double mutant strains thnYthnA3, and thnYthnA1 were generated and complemented with derivatives of pIZ1016 expressing the WT thnY or the mutant alleles, thnY-C40S and thnY-N201G,S206P. In order to analyze these mutations in the natural genomic context, we integrated them into the bacterial chromosome. Therefore, we had to construct a new chromosomal version of the thnY mutation in which the entire gene was replaced by a Km resistance Ω cassette, strain thnY::Ωkm (MPO751), to avoid recombination between thnY sequences. The double-mutant strains, thnY::Ωkm∆thnA3 (MPO752) and the thnY::ΩkmthnA1-D221A (MPO753), were accordingly constructed based on this background strain. For complementation studies, appropriate DNA fragments containing either the wild type thnY allele, the thnY-C40S or the thnY-N201G,S206P derivatives were re-cloned into a promoter-less derivative of pIZ1016, which has just 675 bp of the thnR 3' end and the whole thnY-coding sequence. Theses constructs were designed to place the thnY alleles under the control of the chromosomal Pc promoter. A schematic representation of the genome organization around the thnR and thnY region after the recombination event leading to the integration of the thnY derivatives is shown in the Figure 6A.

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Figure 6. Influence of ThnA3 on the ability of ThnY-C40S and ThnY-N201G,S206P to induce thn expression.

(A) Schematic representation of the genome organization of the thnRY region after the recombination event leading to integration of the thnY derivatives into the chromosome. thnY** denotes WT thnY or its mutant derivatives, while thnR* indicates a deletion of 243 pb from its 5’ end. Arrows indicate the positions and directions of the genes. As shown in the schematic, the P_c promoter drives transcription of thnR and thnY. (B) Ability of ThnY-C40S and ThnY-N201G,S206P to induce thn expression in thnY ::Ωkm (1), thnY::Ωkm∆thnA3 (2) and the thnY::ΩkmthnA1-D221A (3) mutants strains. Plasmids pMPO791 (wild type thnY), pMPO792 (thnY-C40S) and pMPO793 (thnY-N201G,S206P) were used for integration. Cultures were assayed for β-galatosidase activity from a thnC::lacZ reporter fusion after growth under carbon-limiting conditions using tetralin (black bars) and trans-decalin (grey bars) as inducers.

https://doi.org/10.1371/journal.pone.0073910.g006

To study the expression phenotype, the levels of β-galactosidase activity from the thnC::lacZ translational fusion, which was integrated in each strain, was measured after cells were grown with 8 mM of βHB (carbon-limiting conditions) in the presence of either tetralin or trans-decalin. Figure 6B shows that complementation with the WT thnY gene clearly reproduced the expected expression phenotype: (i) complementation of thnY::Ωkm (strain number 1 in the figure) resulted in lower induced levels with trans-decalin than with tetralin; (ii) the levels of thn gene induction were very high in the absence of ThnA3, no matter the inducer molecule, (thnY::Ωkm∆thnA3, strain number 2); and (iii) induction was very low in the genetic background in which reduced ThnA3 is presumably abundant, (thnY::ΩkmthnA1-D221A, strain number 3), regardless of the presence of the inducer. In contrast, ThnY-C40S exhibited the same low level of expression regardless of the inducer molecule, and the genetic background. The absence of ThnA3 (strain 2) or its presence mainly in the reduced form (strain 3) did not substantially affect the expression levels, thus indicating that this mutation renders the protein fully insensitive to the negative effect of the ferredoxin. These results clearly indicate that the information as to whether a molecule is an appropriate inducer or not is transduced from ThnA3 to the regulatory system via the ferredoxin reductase ThnY.

Complementation of the thnY::Ωkm mutant (strain 1 in Figure 6) with thnY-N201G,S206P reproduced what has been shown above in Figures 4A and 5: the thnY-N201G,S206P mutation conferred the inability to discriminate between a weak and a strong inducer. However, tetralin induction increased in the absence of ThnA3 (strain 2) and expression was clearly limited when ThnA3 could not reduce the dioxygenase enzyme (strain 3), regardless of the inducer. These data clearly suggest that ThnY-N201G,S206P is somehow still sensitive to the negative effect of the ferredoxin, although this mutant form does not allow discrimination between the inducers.

Media and bacteria growth conditions

E. coli strains were routinely grown in Luria-Bertani medium at 37 °C; S. macrogolitabida strains were grown at 30 °C in MML rich medium [16] or MM medium [17] with β-hydroxybutyrate as the carbon and energy source.

Antibiotics were used, when were required, at the following final concentrations: ampicillin (100 µg/ml or 5 µg/ml for E. coli or S. macrogolitabida strains respectively); kanamycin (20 µg/ml for E. coli or 10 µg/ml S. macrogolitabida strains); gentamicin (10 µg/ml); and streptomycin (50 µg/ml for S. macrogolitabida strains).

Construction of plasmids and bacterial strains

Strains, plasmids and primers used in this work are summarized in the Table S1. Plasmid pMPO751 bearing thnA3A4RY under the P_tac promoter, was constructed by insertion of a 3.37 kb PvuII-HindIII DNA fragment from pIZ612 into pMPO750 digested with SalI(Klenow) +HindIII.

A 3.35 kb HpaI-XbaI DNA fragment bearing thnRY from pMPO751, was introduced into pIZ652 digested with the same enzymes used to generate pMPO753, which carries an artificial thnA1A2A3A4RY operon. This plasmid was used to create pMPO754. The 6.5 kb XhoI-XbaI DNA fragment bearing the thnA1A2A3A4RY operon from pMPO753 was inserted into the broad-host-range expression vector pIZ1016 digested with SalI-XbaI to obtain pMPO754 in which thnA1A2A3A4RY was transcribed from the P_tac promoter.

Plasmids pMPO756, pMPO757, pMPO763, pMPO764 and pMPO765 carry different mutant thnY alleles under the control of the P_tac promoter were generated by overlapping PCR [18]. To do that, two internal and divergent primers introducing the appropriate mutations were used together with the external primers, and pMPO750 as the template. The two resulting PCR products were mixed in 1:1 and amplified with the external primers to obtain a single DNA segment containing the mutations. Those DNA fragments were cloned into pMPO750 to substitute the thnY wild type region.

pMPO756, which contains the Tyr139Leu (TAT to TTA) substitution in the FAD domain of thnY, was constructed by using thnY-YL1 and thnY-YL2 as internal primers, and SalI-orFY12 and thnY2-1Q as the external ones, respectively. The final 1 kb DNA fragment was digested with SalI+PstI and the 0.49 kb fragment was introduced into pMPO750 digested with the same enzymes.

pMPO757, which contains the Arg137Leu (CGC to CTC) substitution in the FAD domain of thnY, was constructed essentially as pMPO756 but using thnY-RL1 and thnY-RL2 as internal primers. The final PCR fragment was digested and cloned into pMPO750 in the same way that pMPO757.

pMPO763, which contains the Asn201Gly (AAT to GGT) and Ser206Pro (TCG to CCG) substitutions in the NAD(P) H domain of ThnY, was constructed essentially as above but using thnY-NG-SP1 and thnY-NG-SP2 as internal primers. The final 1 kb DNA fragment was digested with SalI+HindIII, and a 0.84 kb DNA fragment was cloned into pMPO750 digested with the same enzymes.

pMPO764, which contains the Cys35Ser (TGC to AGC) substitution in the fer domain of thnY, was executed by using orFY3 and orFY4 as internal primers, and SalI-orFY12 and orFY10 as the external ones, respectively. The final 0.56 kb DNA fragment was digested with SalI+PstI and the 0.49 kb fragment was introduced into pMPO750 digested with the same enzymes.

pMPO765, containing the Cys40Ser (TGC to AGC) substitution in the [2Fe-2S] domain of thnY, was done in the same way that pMPO764 but using orfY7 and orFY8 as internal primers. The final PCR fragment was digested and introduced into pMPO750 as in pMPO764.

Plasmids pMPO791, pMPO792 and pMPO793 harbored the different thnY versions (WT thnY, thnY-C40S and thnY-N201G,S206P respectively) downstream a fragment of 675 pb of the C-terminal region of thnR under no promoter control.

pMPO791, with the wild type thnY was build digesting pIZ1158 with SalI+PstI. The 1.1 kb DNA fragment was cloned into pMPO750 digested with NcoI (T4 endonuclease) +SalI.

pMPO792, which carries the thnY-C40S mutant was done by substitution of the 0.38 kb DNA fragment bearing the mutation of pMPO765 digested with BsaI+PstI, for the same DNA fragment of pMPO791 digested in the same way.

pMPO793 was construct essentially as pMPO791, but the DNA fragment from pIZ1158 digested with SalI+PstI was cloned into pMPO763 NcoI(T4 endonuclease) +SalI.

pIZ1159 was made to generate a chromosomal deletion mutation of thnY. The DNA fragment was made by overlapping PCR using pIZ1158 as template and orFY1+orFY6 as primers to amplify the upstream fragment, and orFY2+orFY5 to amplify the downstream one. Primers orFY1 and orFY2 incorporated an EcoRI restriction site, while orFY5 and orFY6 a SacI one. The resultant PCR products were mixed in 1:1, and amplified with the external orFY1 and orFY2 primers. The final 2.45 kb DNA fragment carrying thnRcopB was digested with EcoRI and cloned into EcoRI-digested Bluescript II SK+ to produce pIZ1157. The Ωkm fragment was obtained by treating pMKm with EcoRI and Klenow, and cloned into the generated SacI site to construct pIZ1159.

The thnY::Ωkm, (ΔthnY) mutant MPO751 was obtained by marker exchange replacement of wild type thnY with the Ωkm fragment present in pIZ1159. A first recombination event leading to integration of pIZ1159 was selected as kanamycin and ampicillin resistant clones. The second recombination event leading to thnY substitution was obtained as ampicillin sensitive kanamycin resistant clones. The MPO752 (thnY::Ωkm,ΔthnA3 double mutant) and MPO753 (thnY::Ωkm,thnA1-D221A double mutant) strains were obtained in a similar way by marker exchange replacement of thnY in the mutant T1031 (ΔthnA3) and T1034 (thnA1’) strains, with the Ωkm of pIZ1159. The authenticity of all of the strains constructed by marker exchange was confirmed by Southern blot hybridation.

To monitor the expression levels of the thn genes by β-galactosidase assays, plasmids carrying thnC::lacZ gene fusions (in pMPO690 for the Δthn mutant strain T690, or in pIZ1002 in all the other cases) were introduced by triparental mating and integrated by homologous recombination.

For complementation analysis, the different plasmids were transferred into the recipient strains and transconjugant colonies were selected on MML agar plates by using resistance to gentamicin, ampicillin, kanamycin and streptomycin. Incorporation of the plasmids and, eventually, their integration into the chromosome, were verified by PCR.

All DNA manipulations were performed according to standard procedures [19]. DNA plasmids were transferred into E. coli and S. macrogolitabida strains by transformation [20] or triparental mating [21] respectively. E. coli DH5α was used as host in all cloning procedures.

Induction assays

Induction assays of S. macrogolitabida strains containing lacZ fusions integrated into the chromosome were performed as previously described [1]. The inducers tetralin, cyclohexane, cis-decalin, trans-decalin and benzene were supplied in the gas phase. When required, IPTG was used at 1 mM final concentration. Cultures were grown for 22 h at 30 °C in minimal medium containing 8 mM βHB as the carbon source, and β-galactosidase activity was assayed as described by Miller [22].

Western Blot Analysis with anti-TFA ThnY antiserum

Whole-cell protein extracts from each TFA strain were prepared from tetralin-induced cultures used for the β-galactosidase assays with (+) and without (-) IPTG. To do that, 8 ml of cultures were harvesting and the cell pellets were resuspended in an appropriate volume of buffer that was adjusted according to differences in optical density at 600 nm. To calculate the concentration of protein and to further ensure that biomass among samples were equivalent, several dilutions in water from these samples were prepared in duplicates and the total amount of protein in each sample was determined by the method of Lowry [23] using bovine serum albumin as the standard. Aliquots of these suspensions were mixed with reducing 2× SDS sample buffer (160 mM Tris-HCl [pH 6.8], 4% [wt/vol] SDS, 20% [vol/vol] glycerol, 0.1% [wt/vol] bromophenol blue, 10% [vol/vol] 2-mercaptoethanol), boiled for 10 min, and centrifuged for 1 min. Extracts corresponding to 250 µg of total protein per sample were run on 12% (w/v) SDS-PAGE polyacrylamide gels [24]. For Western blotting, rabbit anti-ThnY polyclonal antibodies [4] was diluted 5000-fold before use, and the antibody–antigen reaction was visualized using SuperSignal Western Blotting kit (Thermo Scientific) according to the manufacturer’s specifications.