Genetically engineered Acinetobacter sp. strain DF4-8 mopR-like/lux bioreporter was developed to detect phenol through bioluminescence in a concentration range of 2.5 to 100 ppm, demonstrating effectiveness even in aged, contaminated soil (Fig. 1). Its robust response was primarily toward phenol and 3-chlorophenol, with moderate reactions to o-cresol and m-cresol. However, due to the limited sensitivity of strain DF4-8, all other tested phenol derivatives failed to initiate a bioluminescent signal above background levels. The DF4-8 mopR-like promoter/lux construct was also inducible by phenol in E. coli SV17, indicating activity triggered by phenol binding. This mirrored the results obtained by Schirmer et al. [7] regarding the introduction of an A. calcoaceticus NCIB8250 mopR promoter/lacZ fusion into E. coli DH5.
Despite these functional similarities, the putative mopR gene in mopR-like/lux bioreporter showed no significant homology to the mopR gene of A. calcoaceticus NCIB8250. Therefore, this study subjected to identify and characterize the role of this unique mopR-like gene in the phenol degradation pathway of Acinetobacter. The research is centered on the mopR-like gene found in strain DF4-8, focusing on its limited responsiveness to specific phenol derivatives beyond simple phenols. Despite lacking homology to its origin mopR gene from A. calcoaceticus NCIB8250, strain DF4-8 still senses phenol, prompting investigation into the reasons behind this non-similarity. By comparing the sequence of the DF4-8 mopR-like gene, its putative regulatory elements, and promoter activity to a well-characterized strain, the study aims to unravel the mechanisms underlying this differential sensitivity. Additionally, the research delved into the role of conserved motifs, predict the protein structure of the mopR-like gene product, and explore potential regulatory factors influencing its activity.
Motif Discovery and Functional Prediction
The UGENE Open Reading Frame (ORF) search tool was utilized to identify ORFs within the mopR gene region and its clones DF4-8 and DF4-10. Within the mopR gene region, a single ORF, named mopR-ORF, was identified on the positive strand (+). Spanning from nucleotide position 124 to beyond 684, it comprised 561 nucleotides, encoding a putative protein of 186 amino acids. Similarly, within clone DF4-8, an ORF named DF4-ORF8 was located on the positive strand (+). It spanned from nucleotide position 91 to beyond 717, encompassing 627 nucleotides and encoding a putative protein of 208 amino acids. In contrast, clone DF4-10 harbored an ORF named DF4-ORF10 on the negative strand (-). Spanning from nucleotide position 822 to position 376, it had a length of 447 nucleotides and encoded a putative protein of 149 amino acids (Fig. 1). To validate the predicted open reading frames (ORFs) within the mopR gene region and its clones, their entire sequences were used in extensive BLAST searches against both NCBI nucleotide and protein databases. This approach confirmed the accuracy of the ORFs and precisely determined their full sequence lengths.
BPROM analysis identified bacterial promoters within MopR-ORF, DF4-ORF8, and DF4-ORF10 sequences, providing insights into their regulatory mechanisms (Fig. 2). MopR-ORF harbors a single promoter (LDF 3.46) at position 271 flanked by a -10 box (TTTTATGCT) and a -35 box (TTTAAG), potentially regulated by argR (repressor/activator in arginine metabolism) based on identified binding site oligonucleotides TAATTCAT at position 279 with a score of 10. DF4-ORF8 displays a promoter at 588 (LDF 3.39) with a -10 box (AGCTAAAGT), a -35 box (TTGAAA), and predicted phoB (phosphate response regulator) binding site oligonucleotides TTTAATTA at position 534 with a score of 11, DF4-ORF10 possesses two promoters: the first at 81 (LDF 4.01) with a -10 box (TCGTATTAT) and a -35 box (ATGAAA), and the second at 411 (LDF 1.01) with a -10 box (GGGTAACAG) and a -35 box (TTGCGA), with the latter potentially regulated by rpoD17 (RNA polymerase component) based on the identified binding site oligonucleotides TTTTCCTT at position 368 with a score of 7.
The CENSOR analysis was conducted to investigate the associations of transposable elements (TEs) within the sequences of mopR-ORF, DF4-ORF8, and DF4-ORF10 (Fig. 2). Among these sequences, mopR-ORF showed a notable alignment with the BEL-20_ArCe-I transposable element, classified as an LTR Retrotransposon. This alignment covered a sequence of 69 nucleotides, precisely spanning positions 276 to 344 within the mopR-ORF sequence, resulting in an alignment score of 245. Despite displaying a high degree of sequence similarity (0.7714) to its consensus, a single mutation was identified within the BEL-20_ArCe-I element relative to its consensus sequence, occurring at position 341 (GGAT-344), indicating a nucleotide substitution within the aligned region. Conversely, no significant TE associations were found within the sequences of DF4-ORF8 and DF4-ORF10.
Further analysis revealed a masked region within the mopR-ORF sequence, extending from positions 344 to 276, indicating the presence of a repetitive element. Additionally, Repbase annotation provided insights into the characteristics of BEL-20_ArCe-I, including its origin from the filler cichlid genome, a length of 6050 base pairs, and a consensus sequence similarity of approximately 99%. These findings underscore the genomic complexity and evolutionary implications of transposable elements within the examined sequences.
BLAST analysis of the intergenic spacer (iS) region using the ISfinder_Nucl database revealed significant matches between the query sequences and insertion sequence (IS) elements (Fig. 2). The mopR-ORF sequence (561 bp) exhibited a perfect match (40.1 bits and 100% identity) with ISSgl1 (IS5 family) from Sodalis glossinidius. The complete alignment spanning positions 256–275 of mopR-ORF and 399 − 380 of ISSgl1 suggests the mopR-ORF sequence is likely identical to the ISSgl1 transposon. For the DF4-ORF8 DNA sequence (627 bp), a strong match (34.2 bits) was identified with ISSme2 (IS21 family) from Sinorhizobium medicae. Within a specific region (positions 189–205 of DF4-ORF8 aligning with positions 1272–1288 of ISSme2), the sequences displayed perfect identity (17/17 nucleotides), suggesting a close evolutionary relationship or origin from ISSme2. Finally, the DF4-ORF10 sequence (447 bp) shared 95% identity (19/20 nucleotides) with ISHahy5 (IS3 family) from Halanaerobium hydrogeniformans across aligned positions (340–359 of DF4-ORF10 and 110–129 of ISHahy5), resulting in a strong match (32.2 bits). This finding indicates a potential evolutionary link between DF4-ORF10 and ISHahy5.
Protein Structure Prediction and Analysis
Utilizing the MUSCLE tool in UGENE, multiple sequence alignments were conducted for mopR-ORF, DF4-ORF8, and DF4-ORF10 (Fig. 3). The mopR-ORF features diverse amino acids including Leucine, Serine, Arginine, and Glutamine, with repetitive regions "QL" (two times) and "AE" (seven times). DF4-ORF8 displays repetitive stretches "KWQATKVGV" at positions 81–89 and 101–112, and "GVLGAGMMG" at positions 88–96 and 111–119, alongside Leucine, Alanine, Glycine, and Valine. DF4-ORF10 lacks repetitive motifs but contains Methionine, Isoleucine, Lysine, and Aspartic acid, showing variability in length and composition. To validate identified repeated regions within DF4-ORF8 sequence, NCBI Blast search was performed. The "KWQATKVGV" repeat sequence of DF4-ORF8 aligned significantly with 3-hydroxyacyl-CoA dehydrogenase NAD-binding domain-containing proteins from Gordonia otitidis, Acinetobacter ursingii, and Acinetobacter venetianus, scoring 32.0 with 100% coverage and identity. Similarly, "GVLGAGMMG" displayed significant alignments with sequences from Abscondita terminalis, fatty oxidation protein fadB from Mycobacterium tuberculosis, and 3-hydroxyacyl-CoA dehydrogenase from Betaproteobacteria bacterium, scoring 31.2 with 100% coverage and identity.
Additionally, in a quest to elucidate the functional roles of specific regions within three translated proteins, GLAM2 analysis within the MEME Suite identified conserved motifs; mopR-ORF (positions 158–171), DF4-ORF8 (positions 156–169), and in DF4-ORF10 (positions 117–130). Subsequent validation using BLAST searches provided compelling evidence for their functional significance (Fig. 4). For mopR-ORF, BLAST searches revealed significant alignments with proteins annotated as phenol degradation transcriptional regulators (MopR), sigma 54-interacting transcriptional regulator and XylR N-terminal domain-containing protein from diverse Acinetobacter strains. Notably, these alignments exhibited exceptionally low E-values (below 10^-5), highlighting a highly conserved role for MopR. This suggests a critical function for the identified motif (positions 158–171) in regulating phenol degradation pathways across various Acinetobacter species.
Similarly, BLASTp searches confirmed a highly conserved motif within DF4-ORF8 (positions 156–169). Closely matching proteins across Acinetobacter species displayed 100% identity and an E-value of 6e-04, further substantiating the motif's significance. Importantly, these matching proteins were functionally linked to fatty acid metabolism, in particular 3-hydroxyacyl-CoA dehydrogenase NAD-binding domain-containing protein, suggesting a potential role for DF4-ORF8 in regulating this metabolic process. Finally, BLASTP searches for DF4-ORF10 identified significant alignments with SDR family oxidoreductase and 3-hydroxybutyrate dehydrogenases from a range of Acinetobacter species. These alignments displayed a high degree of sequence similarity (E-values ranging from 2e-04 to 5e-04) and 100% identity. This strong conservation suggests a crucial role for the DF4-ORF10 motif (positions 117–130) in modulating the enzymatic activity of 3-hydroxybutyrate dehydrogenases within Acinetobacter species.
InterProScan analysis of mopR-ORF revealed a multifaceted protein potentially involved in aromatic catabolism, cellular redox processes, ligand binding, and transport (Fig. 5A). The N-terminal domain (residues 1–78) suggests a role in regulating genes for aromatic compound breakdown due to its homology to "activators of aromatic catabolism". The central domain (residues 91–153), designated as "V4R_2_a", is associated with reductases but might have diverged in function within mopR-ORF. Additionally, a "ligand-binding domain" (residues 15–148) suggests potential ligand interactions, though the specific ligand and its function remain elusive. Furthermore, mopR-ORF was identified as a "trafficking protein particle complex subunit 3", hinting at its involvement in cellular transport processes.
The analysis of DF4-ORF8 identified a single domain spanning residues 99–206, encompassing the entire protein (Fig. 5B). This domain is designated as "3-hydroxyacyl-CoA dehydrogenase, NAD binding domain" (IPR006176, PF02737) and belongs to the "NAD(P)-binding Rossmann-fold superfamily" (IPR036291). This domain assignment suggests DF4-ORF8's function in fatty acid metabolism (GO:0006631) and its ability to bind NAD+ (GO:0070403), a crucial cofactor for its enzymatic activity. Additionally, the protein was identified as a "trifunctional enzyme subunit alpha" (PTHR43612), further supporting its involvement in fatty acid metabolism.
DF4-ORF10 protein analysis possesses multiple conserved short-chain dehydrogenase/reductase (SDR) domains, indicative of its enzymatic function (Fig. 5C). These domains encompass residues 16–32, 42–61, 63–80, and 108–128, and harbor a conserved catalytic site (residues 29–57, SINGLIGFAGKAGYNSAKHGVIGLTKVAA) specific to SDRs. Furthermore, an NAD(P)-binding domain spanning residues 2-131 is predicted, crucial for SDR activity as NAD(P) acts as a cofactor. Residues 42 (Y), 46 (K), and 72–77 (PGYVDT) are particularly important for catalysis and cofactor binding. Functionally, DF4-ORF10 belongs to the oxidoreductase class (GO:0016491), confirming its role in oxidation-reduction reactions.
Phyre analysis of mopR-ORF identified a primary template (c7vqfA_) showing remarkable sequence similarity (100% identity, 99% coverage) to the protein MopR (PDB ID: 7vqf) from Acinetobacter calcoaceticus, known as a phenol sensing regulator (Fig. 6A). The structural data from the Protein Data Bank Europe (PDBe) aligns with domains associated with regulating aromatic catabolism in MopR, supporting Phyre's prediction for mopR-ORF. This structure, resolved by X-ray diffraction at 2.3 Å resolution, illustrates a single protein chain (229 amino acids) potentially interacting with inositol hexaphosphate, acetate, and zinc ions. The high sequence similarity between MopR and mopR-ORF suggests potential involvement in phenol sensing and aromatic compound catabolism for mopR-ORF.
The analysis of DF4-ORF8 identified a template from Mycobacterium tuberculosis 3-hydroxyacyl-CoA dehydrogenase (PDB 7o4t) with 100% identity and 89% coverage (Fig. 6B). PDB 7o4t offers insights into a Mycobacterium tuberculosis beta-oxidation trifunctional enzyme complex, integrating enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and thiolase activities within a single structure. The complex contains bound molecules like Coenzyme A (an essential cofactor), glycerol, and phosphate (likely from crystallization buffer), along with sulfate (possibly contributing to stability).
The Phyre2 analysis confidently predicted that the translated protein DF4-ORF10, consisting of 149 residues, adopts the structure of a 3-hydroxybutyrate dehydrogenase enzyme from Acinetobacter baumannii (Fig. 6C). This prediction showed a high level of confidence with 95% sequence coverage and 99.95% confidence (142 out of 194 residues). The predicted structure perfectly aligns with the top-scoring template (PDB ID: c6zzsB_) retrieved from the Protein Data Bank (PDB). The template is annotated as an oxidoreductase in the PDB header and corresponds to a crystal structure of 3-hydroxybutyrate dehydrogenase complexes with NAD + and 3-oxovalerate. Notably, DF4-ORF10 and c6zzsB_ exhibit a significant 91% identity over 142 aligned residues, indicating a close evolutionary relationship or possible shared function between the two sequences.
The bioinformatics tools developed by the DTU Health Tech Department of Health Technology were used to analyze the Rossmann folds and binding specificities of FAD, NAD, and NADP cofactors using the Cofactory online tool in three proteins: mopR-ORF, DF4-ORF8, and DF4-ORF10. The analysis revealed that DF4-ORF8 contains two domains: the first domain (QATKVGVLGAGMMGADVTKW, positions 76–95) lacks predicted cofactor binding, while the second domain (QATKVGVLGAGMMGADVTKWQATKVGVLGAGMMGA, positions 96–138) shows specificity for NAD. The cofactors analysis of DF4-ORF10 revealed a domain with predicted binding affinity to cofactors FAD, NAD, and NADP, with respective probabilities of 0.004, 0.078, and 0.046. However, no specific cofactor was identified in the sequence. The sequence spans from position 48 to 73 and is represented by the amino acid sequence (GVIGLTKVAALECARDGITVNALCPG). No predictions were made for the third sequence, mopR-ORF. This prediction was supported by significant sequence similarity (80.5% − 84.7% identity) observed through NCBI BLAST between DF4-ORF8 domain 2 and NAD-binding domains found in 3-hydroxyacyl-CoA dehydrogenase enzymes from various bacterial species, particularly Acinetobacter.
In terms of free radical scavenging (FRS) and chelation (CHEL) activities, peptides with predicted functionalities were found in all three sequences. Peptides from DF4-ORF10 displayed the highest FRS scores (around 0.63), including sequences like "RPSCCYGWW" and "NRPSCCYGWW," suggesting their potential role in mitigating cellular damage caused by free radicals. Similarly, several peptides from DF4-ORF8 exhibited high FRS scores (around 0.62), indicating a possible role in free radical scavenging. While mopR-ORF showed moderate FRS scores (around 0.60), it possessed the highest CHEL scores (around 0.31) for peptides containing sequences like "NELHLSHDLK." These CHEL peptides might have the potential to bind metal ions, potentially regulating their availability for unwanted reactions within the cell.
Using NetPhosBac 1.0a for predicting protein phosphorylation sites, potential candidates were identified across all three protein sequences (DF4-ORF8, DF4-ORF10, and mopR-ORF). While all proteins exhibited predicted sites with scores ranging from 0.115 to 0.644, indicating varying confidence levels, only DF4-ORF8 (positions 36, 67, and 149) and DF4-ORF10 (positions 53, 66, and 147) displayed three sites exceeding the 0.5 threshold in NetPhosBac 1.0a. This threshold signifies a stronger prediction for phosphorylation at these locations. MopR-ORF had two sites (positions 80 and 95) exceeding the 0.5 threshold.