**3. Results**

#### *3.1. Engineered CatM Variants: Amino Acid Replacements at Positions 160 and 293*

In an effort to make CatM respond to benzoate, *A. baylyi* strains were engineered to encode CatM (H160R), CatM (F293Y), or CatM (H160R, F293Y) (designated ACN662, ACN682, and ACN685, Table S1). These amino acid replacements were designed to match residues in BenM-EBD that interact with benzoate (Figure S2). To ensure that transcriptional regulation could be attributable to the CatM variants, *benM* was also disrupted. The engineered strains all grew on anthranilate, catechol, and muconate as sole carbon sources. Such growth requires transcription from the *cat*-operon promoter, PcatB (Figure 1), indicating that the variants are functional as *cat*-gene regulators. To assess PbenA regulation, growth on benzoate was evaluated. Without BenM, neither wild-type CatM, in strain ISA36 [3] nor CatM (F293Y) in ACN682 supported growth on benzoate as the carbon source. In contrast, CatM (H160R) in ACN662, and CatM (H160R, F293Y), in ACN685, conferred growth on benzoate, suggesting that H160R enables higher than normal levels of PbenA transcription.

#### *3.2. Transcriptional Regulation of PbenA by CatM Variants*

A *benA*::*lacZ* fusion was used to replace *benA*, thereby preventing growth on benzoate as the carbon source and enabling benzoate to be tested as a non-metabolized effector [3]. β-Galactosidase (LacZ) activity reflects PbenA regulation by CatM(F293Y), CatM(H160R), CatM(H160R,F293Y), or wild-type CatM in strains ACN717, ACN673, ACN694, and ACN1307, respectively (Figure 3). BenM is the major regulator of PbenA. Without effectors, a tetramer of BenM or CatM, can bind Site 1 and Site 3 to repress transcription of *benA* or *catB* (Figure 3C). Effectors cause a shift in which protein binding to Sites 1 and 2 improves RNAP access to Site 3 [2]. Despite possible cross-regulation, the maximum level of CatM-activated transcription was 18-fold lower than for BenM (ACN1307 versus ACN1232). However, the H160R replacement increased this low-level CatM-mediated response to muconate (ACN673 and ACN694). The elevated transcription levels remained significantly below that for BenM (ACN1232), ye<sup>t</sup> they are comparable to those of other CatM variants that enable BenM-independent growth on benzoate [4]. Regulation by CatM(F293Y), which did not permit growth on benzoate, was comparable to wild-type CatM, which also fails to allow such growth.

While the *catM* changes failed to recapitulate the effects of benzoate with BenM, this failure might be due to problems interacting with PbenA DNA as opposed to problems with binding benzoate. In previous studies, CatM activated higher levels of *benA* transcription when there was a transversion (T-to-A) at position −40 relative to the transcriptional start site [3,4]. Here we tested regulation of this promoter (PbenA5146) by CatM and its variants CatM(F293Y), CatM(H160R), and CatM(H160R,F293Y) using the *lacZ* fusion (in ACN157, ACN827, ACN832, and ACN839, respectively). CatM yielded higher levels of transcription from PbenA5146 than PbenA under all conditions (ACN157 versus ACN1307, Figure 3). At this promoter, the variants all increased transcription even further than wild-type CatM. Benzoate led to expression levels 170% or 155% of the non-induced levels for CatM(H160R) or CatM(H160R,F293Y), respectively (Figure 3). Despite this suggestion of a minor response to benzoate, muconate-mediated transcription was inhibited by benzoate. In contrast, for BenM, benzoate works synergistically with muconate (ACN1232) [2].

**Figure 3.** Relative expression of LacZ. Transcription was controlled by PbenA, or, PbenA5146, which differs by a mutation at −40 relative to the *benA* transcriptional start site. (**A**) Enlarged scale displays basal activity (no added effectors). (**B**) Effectors were added (or not) as indicated. ACN1232 encodes BenM but not CatM, and ACN1307 encodes CatM but not BenM. Other strains encode a CatM variant. Cultures were grown in LB. LacZ activity is reported relative to uninduced ACN1232 (2.6 ± 0.51 nmol/min/mL/OD600). Activities are averages of at least four repetitions; standard deviations were <20% of the average value. (**C**) Identical nucleotides in aligned PbenA and PcatB regions are indicated (:). The transcriptional start site (+1) and promoter (−10 and −35 regions) are shown for *catB*. For both regions, Site 1 matches the consensus LTTR-binding motif (T-N11-A, within dyad symmetry, ATAC-N7-GTAT). Site 2 and Site 3 differ slightly from this consensus. The PbenA5146 mutation (T) is marked.

#### *3.3. Spontaneous Mutant with Increased ben-Gene Expression: Changesne in the CatM-DBD*

A different approach was used in another attempt to isolate a benzoate-responsive CatM. While ACN682, encoding CatM(F293Y), does not grow on benzoate, its *catM* mutation might facilitate the ability of additional mutations to confer this trait. Based on this rationale, Ben<sup>+</sup> colonies were directly selected from ACN682, as was done previously for wild-type CatM [3,27]. One ACN682-derived mutant encoded two changes, F293Y and I18F. To test the role of I18F, a strain was made to encode only this change which matches residue 18 of BenM-DBD (Figure 2). CatM(I18F) conferred Ben<sup>+</sup> growth (ACN1095), indicating increased transcription of PbenA. When tested with the *benA*::*lacZ* fusion in ACN1111, there was no response to benzoate as a sole effector, and benzoate decreased the muconate-inducible expression (data not shown). It appears that the Ben<sup>+</sup> phenotype arises from an increase in muconate-induced PbenA expression (ACN1111 compared to ACN1307; Figure 4).

**Figure 4.** Relative LacZ activity of a chromosomal *benA*::*lacZ* fusion. The only strain encoding BenM is ACN1232, which has no CatM. The dotted line shows the level of the CatM response to muconate (ACN1307). Cultures were grown on pyruvate or muconate as the sole carbon source. Expression is reported relative to the basal level of ACN1232 (6 ± 2 nmol/min/mL/OD600). Values represent the average of at least three independent replicates. Standard deviations were <20% of the average value.

Since I18F makes the variant more like BenM, all nine DBD differences were considered (Figure 2). Residue 38, in the recognition helix of the helix-turn-helix (HTH) DNA-binding motif, is implicated in BenM-DNA interactions [23]. Therefore, strains were made to encode CatM(K38N) and CatM(I18F, K38N) (ACN1193 and ACN1249, respectively). Both strains grew on anthranilate, muconate, and benzoate, indicating that the CatM variants are functional. Muconate enabled CatM(K38N) and CatM(I18F, K38N) to activate higher levels of PbenA transcription than CatM (ACN1194, ACN1251 versus ACN1307, Figure 4). For the double-replacement variant, gene expression was comparable to that mediated by BenM (in ACN1232). CatM(I18F, K38N) also resulted in high PbenA basal expression (Figures 3 and 4). However, there was no transcriptional activation in response to benzoate. Moreover, for both variants with K38N, benzoate decreased the ability of muconate to activate transcription (ACN1251 in Figure 3).

#### *3.4. Further Investigation of DBD Residues in BenM and CatM*

The ability of BenM to regulate PbenA might be weakened by having the residues of CatM at positions 18 and 38. To test this possibility, strains were made to encode BenM(F18I,N38K). When tested with a *benA*::*lacZ* reporter, regulation by this variant was significantly decreased under all conditions relative to BenM. Nevertheless, BenM(F18I,N38K) remained capable of activating transcription synergistically in response to benzoate and muconate (Supplementary Figure S3).

The entire DBD of CatM was replaced with that of BenM by changing nine amino acids. This variant (BenM-DBDCatM) enabled growth on muconate, anthranilate, and benzoate as sole carbon sources (ACN1234). When regulating a *benA*::*lacZ* reporter, this variant activated transcription similarly to BenM with muconate (ACN1239 versus ACN1232). However, there was no response to benzoate as the sole effector, and benzoate decreased muconate-inducible expression as observed for the previously studied CatM variants (Figure 3).

#### *3.5. Benzoate-Responsive CatM Variants Obtained with Combinations of DBD and EBD Changes*

Starting with BenM-DBDCatM, we altered the EBD in further attempts to create a response to benzoate. BenM-DBDCatM (H160R, F293Y) enabled growth on benzoate (ACN1301, Table 1). This variant (in ACN1302) also enabled muconate or benzoate to activate high-level transcription (Figures 3 and 4). Notably, there was a BenM-like regulatory pattern when benzoate and muconate were added together. This pattern indicated that 11 amino acid changes are sufficient for a benzoate-responsive CatM capable of synergistic transcriptional activation.


**Table 1.** Growth on Benzoate as the Sole Carbon Source a.

a Strains had comparable growth rates with succinate as the sole carbon source (data not shown); b Averages of at least four determinations; c Time between inoculation and start of exponential growth.

Another combination of changes, CatM(I18F,K38N,H160R,F293Y), generated a Ben<sup>+</sup> strain (ACN1345, Table 1). While two DBD changes enabled muconate-inducible expression, the two additional EBD changes (in ACN1347) were required for induction by benzoate (a 5.5-fold increase in expression, Figure 3). Moreover, when benzoate and muconate were both provided, gene expression increased for CatM(I18F,K38N,H160R,F293Y). In contrast, for CatM(I18F,K38N), benzoate inhibited the response to muconate, Figure 3.

#### *3.6. Promoter Specificity and Regulator-DNA Binding A*ffi*nities*

We also studied PcatB. While CatM-DBD should recognize this region, CatM(H160R,F293Y) did not activate transcription of a *catB*::*lacZ* fusion in response to benzoate (ACN1389, Figure 5). With BenM, PcatB transcription was high, but the pattern of effector responses differed from what occurred at PbenA. Notably, benzoate failed to enhance transcriptional activation in response to muconate (ACN1375, Figure 5). When the BenM-EBD was combined with the CatM-DBD, the overall response to effectors was lessened and the response pattern was altered (ACN1367 compared to ACN1375, Figure 5). As observed at PbenA, a combination of DBD and EBD changes enabled a response to benzoate. Two CatM variants increased gene expression from PcatB in response to benzoate, BenM-DBDCatM(F293Y,H160R) in ACN1369 and CatM(I18F,K38N,H160R,F293Y) in ACN1393, Figure 5. With these variants, benzoate enhanced transcriptional activation by muconate.

To evaluate binding to the operator–promoter regions, an electrophoretic mobility gel shift assay (EMSA) was used. However, because the DNA in the assay includes all three regulatory binding sites (Figure 3), the measured Kd coefficients do not distinguish between repression and activation. Thus, the affinity of BenM for PbenA, appears to be of the same order of magnitude regardless of the presence of effectors (Table 2). CatM appeared to have a slightly higher Kd, which corresponds to a lower affinity, for this DNA. Interestingly, the CatM variants that carry the DBD of BenM have a higher affinity for PbenA than either CatM or BenM. This increased affinity appears to require more than the two amino acid changes in the HTH region of the DBD (I18F and K38N). Moreover, increased affinity does not correlate with benzoate responsiveness. These results sugges<sup>t</sup> the hybrid proteins have a conformation that differs from those of the wild-type CatM or BenM.

**Figure 5.** Expression from a chromosomal *catB*::*lacZ* transcriptional reporter. ACN1375 encodes BenM but not CatM. All other strains lack BenM. All have a *benA* disruption to prevent benzoate catabolism. Cultures were grown on 20 mM pyruvate with or without added effectors. β-Galactosidase (LacZ) activity is reported relative to uninduced ACN1366 (0.71 ± 0.5 nmol/min/mL/OD600). Activities are the average of at least three repetitions, and standard deviations were <10% of the average value.


a Values represent averages of four replicates with *p* > 0.001.

In experiments with PcatB, CatM had a higher affinity than BenM for this DNA (Table 2). As for the PbenA region, the CatM variants with the entire BenM-DBD region had a higher affinity for the promoter DNA than did the variant with two replacements in the DBD. While it is difficult to infer the significance of the variations in Kd values, there appears to be no correlation between the ability of a CatM variant to respond to benzoate and its binding affinity (high or low) for PbenA or PcatB.

#### *3.7. Regulator-DNA Interactions at PbenA: A Structural Model with RNAP*

To understand protein-DNA interactions better, a model was built with available structures of BenM-DBD bound to PbenA and PcatB and *E. coli* RNAP (Figure 6) [23,24]. In this model, Site 2 overlaps the −35 region of the promoter. A run of adenosine nucleotides within Site 2, with a T nucleotide in the middle, appears to create a promoter feature that may increase transcription by interacting with the RNAP α-subunits, an UP element. The relative position of BenM-DBD to the promoter is typical of a class II σ70 promoter in which a regulator directly contacts domain 4 of the sigma factor of bound RNAP to stabilize the initiation complex [28]. However, the model suggests that the BenM-DBDs would not directly contact σ70. The EBD (not in the model) could make contacts with the sigma factor, but the bulk of the EBD units must sit on the opposite face of the RNAP if BenM

is a tetramer. In contrast, for class I σ70 promoters, regulators do contact the α-subunit C-terminal domains (α-CTD), but typically the regulatory proteins bind further upstream of bound RNAP. Protein surface residues of the DBD-dimers that flank the UP-element, such as F31 of GcvA (C26 of BenM), have been implicated in α-CTD interactions [29]. The conserved −35 σ70 recognition-sequence in PbenA (TTGAAC vs. consensus TTGACA) suggests that de-repression by conformational change from Site 1 and Site 3 to Site 1 and Site 2 may be a significant aspect of transcriptional activation along with UP-element interactions.

**Figure 6.** Structural model of BenM-PbenA interactions. (**A**). PbenA with binding sites for BenM (underlined). A potential UP element [30] is indicated and discussed in the text. The boxed nucleotide (T) corresponds to the mutation in PbenA5146. (**B**). Model of the initiation complex at PbenA rendered as a ribbon representation. BenM-DBD subunits are colored pale green, dark magenta, gold, and magenta going from the 5' (left) to 3' of the *benA* promoter; RNA polymerase (RNAP) subunits are green and cyan (α), salmon (β), grey (β'), blue (ω) and yellow (σ).
