Introduction

The increasing demand for sustainable agricultural practices has intensified the search for environmentally friendly alternatives to chemical fertilizers and synthetic plant growth regulators. Excessive application of agrochemicals has contributed to soil degradation, loss of microbial diversity, environmental pollution, and declining soil fertility, posing significant challenges to global food security [1]. Consequently, plant growth-promoting bacteria (PGPB) have emerged as an attractive biological alternative due to their ability to enhance plant growth, improve nutrient acquisition, alleviate abiotic stress, and suppress plant pathogens through diverse biochemical and physiological mechanisms. These beneficial microorganisms colonize the rhizosphere, rhizoplane, or internal plant tissues and establish mutually beneficial interactions with host plants, ultimately contributing to improved crop productivity while reducing dependence on chemical inputs [2].

Plant growth-promoting bacteria facilitate plant development through both direct and indirect mechanisms. Direct mechanisms include biological nitrogen fixation, phosphate solubilization, potassium mobilization, siderophore production, synthesis of phytohormones, and improvement of mineral nutrient availability [3]. Indirect mechanisms involve the suppression of phytopathogens through the production of antibiotics, hydrogen cyanide, hydrolytic enzymes, volatile organic compounds, and competitive exclusion, as well as the induction of systemic resistance in plants. Among these diverse mechanisms, microbial production of phytohormones, particularly indole-3-acetic acid (IAA), has received considerable attention because of its central role in regulating plant growth and development [4].

Indole-3-acetic acid is the most abundant naturally occurring auxin and serves as one of the principal signaling molecules governing numerous physiological and developmental processes in higher plants. IAA regulates cell elongation, cell division, tissue differentiation, vascular development, apical dominance, lateral root initiation, adventitious root formation, phototropism, gravitropism, and fruit development. Furthermore, microbial IAA significantly influences root system architecture by stimulating the formation of root hairs and lateral roots, thereby increasing the effective root surface area available for water and nutrient uptake [5]. Enhanced root development consequently improves plant vigor, nutrient acquisition efficiency, and tolerance to environmental stresses such as drought, salinity, and heavy metal toxicity. For these reasons, IAA-producing bacteria have become important components of microbial biofertilizers and sustainable crop management strategies [6].

Unlike plants, bacteria possess remarkable metabolic diversity and are capable of synthesizing IAA through several independent biosynthetic pathways. Most bacterial pathways are dependent on the amino acid L-tryptophan, which is commonly released into the rhizosphere through root exudates [7]. The principal tryptophan-dependent pathways include the indole-3-pyruvate (IPyA) pathway, the indole-3-acetamide (IAM) pathway, the tryptamine (TAM) pathway, the indole-3-acetonitrile (IAN) pathway, and the tryptophan side-chain oxidase pathway. Among these, the IPyA pathway is considered one of the most widespread in plant-associated bacteria and is primarily mediated by the enzyme indole-3-pyruvate decarboxylase, encoded by the ipdC gene [8]. Other important enzymes involved in alternative pathways include tryptophan monooxygenase (iaaM), indoleacetamide hydrolase (iaaH), aldehyde dehydrogenases, aminotransferases, and several auxiliary metabolic enzymes. The diversity of these biosynthetic pathways enables different bacterial species to synthesize IAA under varying environmental conditions and contributes to the wide variability observed in auxin production among bacterial taxa [9].

Among the diverse groups of plant growth-promoting bacteria (PGPB), members of the genera Pseudomonas, Serratia, and Bacillus have been extensively investigated because of their remarkable metabolic diversity and their ability to enhance plant growth through multiple direct and indirect mechanisms [10]. These bacteria efficiently colonize the rhizosphere and establish beneficial interactions with plants by improving nutrient availability, producing phytohormones, suppressing phytopathogens, and enhancing plant tolerance to environmental stresses. Owing to these multifunctional characteristics, they have become important components of sustainable agricultural systems and microbial biofertilizer formulations [11].

Species belonging to the genus Pseudomonas are recognized as one of the most metabolically versatile groups of Gram-negative bacteria inhabiting soil and plant-associated environments. They possess exceptional adaptability to diverse ecological niches and are capable of producing a wide range of secondary metabolites that contribute to plant growth promotion. Numerous studies have demonstrated that several Pseudomonas species synthesize indole-3-acetic acid through tryptophan-dependent biosynthetic pathways, thereby stimulating root elongation, lateral root development, and nutrient uptake [12]. In addition to auxin production, these bacteria produce siderophores, biosurfactants, antibiotics, hydrogen cyanide, and extracellular enzymes that collectively improve plant health and suppress soil-borne pathogens. Their relatively large genomes encode numerous regulatory and metabolic pathways, making Pseudomonas an excellent model for investigating microbial mechanisms involved in plant growth promotion [13].

Members of the genus Serratia have also gained increasing attention as beneficial plant-associated bacteria because of their ability to produce multiple growth-promoting metabolites. Several environmental isolates have been reported to synthesize indole-3-acetic acid, solubilize inorganic phosphate, produce siderophores, and secrete extracellular hydrolytic enzymes that facilitate nutrient cycling and biological control of phytopathogens [14]. Furthermore, Serratia species exhibit considerable physiological adaptability and can successfully colonize the rhizosphere of numerous crops. Comparative genomic studies have revealed genes associated with phytohormone biosynthesis, stress tolerance, nutrient acquisition, and secretion systems, suggesting their potential for applications in sustainable agriculture and environmental biotechnology [15].

The genus Bacillus represents one of the most widely exploited groups of beneficial bacteria in agricultural biotechnology because of its ability to produce environmentally resistant endospores that ensure long-term survival under adverse conditions [16]. Numerous Bacillus species have been reported to synthesize indole-3-acetic acid together with a variety of extracellular enzymes, antimicrobial peptides, lipopeptides, volatile organic compounds, and other bioactive metabolites that contribute to plant growth promotion and disease suppression [17]. Their excellent environmental stability, ease of large-scale cultivation, and long history of safe agricultural use have resulted in the development of several commercial microbial inoculants based on Bacillus strains. In addition, the availability of complete genome sequences has facilitated detailed investigations into the molecular mechanisms governing phytohormone biosynthesis, stress adaptation, and plant–microbe interactions [18].

Although these bacterial genera are individually recognized as potential producers of indole-3-acetic acid, considerable variability exists among species and strains with respect to the quantity of auxin produced and the biosynthetic pathways involved. Such variation may arise from differences in genomic composition, regulation of tryptophan metabolism, enzyme activity, environmental conditions, and nutrient availability. Therefore, comparative evaluation of representative bacterial species under identical culture conditions provides valuable insights into their relative auxin-producing potential and establishes a foundation for correlating phenotypic observations with underlying genetic determinants [19].

In the present investigation, Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis were selected as representative bacterial species because of their well-documented metabolic capabilities, availability of complete genome sequences, and reported ability to synthesize plant growth-promoting metabolites. Instead of isolating bacteria from rhizosphere soil, authenticated laboratory cultures were employed to eliminate variability associated with environmental isolates and to enable standardized comparative analysis under controlled experimental conditions. The use of reference cultures further facilitated integration of phenotypic observations obtained through broth- and agar-based Salkowski assays with genome-based investigations of auxin biosynthetic pathways, thereby providing a comprehensive understanding of the molecular basis of indole-3-acetic acid production in these bacteria.

Accurate detection of microbial indole-3-acetic acid (IAA) production is an essential step in the identification and characterization of plant growth-promoting bacteria. Over the past several decades, a variety of analytical techniques have been developed for IAA estimation, including high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis, enzyme-linked immunosorbent assays (ELISA), and colorimetric methods. Although chromatographic and spectrometric techniques provide high sensitivity, specificity, and accurate quantification, they require sophisticated instrumentation, skilled personnel, and relatively high operational costs, limiting their routine application in many microbiology laboratories. Consequently, rapid colorimetric assays continue to serve as valuable preliminary screening tools for identifying potential IAA-producing microorganisms [20] [21].

Among the available colorimetric methods, the Salkowski assay remains the most widely employed qualitative screening technique because of its simplicity, rapidity, low cost, and ease of implementation. The assay is based on the reaction between indolic compounds and ferric ions in a strongly acidic medium, resulting in the development of characteristic yellow, orange, pink, or reddish coloration depending on the concentration and composition of indole derivatives present in the culture filtrate. The intensity of the developed colour provides an initial indication of IAA production and enables rapid comparison of multiple bacterial isolates or cultures under identical experimental conditions. Owing to these practical advantages, the Salkowski assay has become a standard preliminary method for screening plant growth-promoting bacteria in agricultural and environmental microbiology [22].

In addition to broth-based detection, agar-based Salkowski assays provide a convenient approach for visualizing auxin-producing colonies directly on solid media. Following bacterial growth on tryptophan-supplemented agar, application of Salkowski reagent allows localized colour development around colonies capable of producing indolic compounds. This method facilitates rapid differentiation of positive colonies without the need for liquid culture processing and is particularly useful during primary screening of large numbers of bacterial isolates. When broth- and agar-based assays are employed together, they provide complementary qualitative information regarding the auxin-producing capability of bacterial cultures and improve confidence in preliminary screening results [23].

Despite its widespread use, the Salkowski assay possesses several inherent limitations that should be considered during interpretation of results. The reagent reacts with a range of indole-containing metabolites rather than exclusively with indole-3-acetic acid, potentially leading to overestimation or false-positive observations in certain bacterial species [24]. Furthermore, colour intensity may be influenced by culture composition, incubation conditions, tryptophan availability, pH, and the accumulation of intermediate metabolites associated with alternative biosynthetic pathways. Consequently, the assay is generally regarded as a qualitative or semi-quantitative screening technique rather than a definitive method for precise IAA quantification. Nevertheless, when performed under standardized experimental conditions and interpreted alongside complementary biological evidence, the Salkowski assay remains an effective and reliable approach for comparative evaluation of microbial auxin production [25].

Recent advances in microbial genomics and computational biology have substantially expanded our understanding of bacterial phytohormone biosynthesis. The rapid increase in publicly available whole-genome sequences has enabled comprehensive identification of genes involved in tryptophan metabolism and auxin biosynthetic pathways [26]. Comparative genomic analyses can reveal the distribution of key biosynthetic genes, predict metabolic capabilities, identify conserved catalytic domains, and provide insights into the evolutionary relationships among IAA-producing bacteria. Functional annotation of these genes further facilitates prediction of enzyme activity and pathway organization, allowing phenotypic observations to be interpreted within a molecular framework [27].

Protein-level bioinformatics has further enhanced the investigation of microbial metabolic pathways by enabling comparative sequence analysis, conserved motif identification, structural modelling, protein–protein interaction prediction, and molecular docking studies [28]. These computational approaches provide valuable information regarding enzyme architecture, substrate-binding characteristics, catalytic residues, and evolutionary conservation, thereby improving our understanding of the molecular mechanisms governing auxin biosynthesis. Integration of phenotypic screening with genome-based and structural bioinformatics therefore represents a comprehensive strategy for investigating microbial IAA production beyond conventional laboratory observations [29].

Although numerous studies have independently reported either experimental screening of IAA-producing bacteria or computational analyses of auxin biosynthetic genes, investigations integrating qualitative phenotypic detection with comparative genome-based characterization of representative plant growth-promoting bacteria remain relatively limited [30]. Establishing relationships between observable auxin-producing phenotypes and the underlying genetic determinants can improve functional interpretation of microbial plant growth-promoting traits and provide valuable information for selecting bacterial strains with enhanced agricultural potential. Accordingly, the present study combines conventional broth- and agar-based Salkowski assays with comparative genomic and protein-based analyses to investigate the auxin biosynthetic potential of Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis [31]. By integrating experimental observations with genome-derived functional predictions, the study aims to provide a broader understanding of the molecular basis of bacterial indole-3-acetic acid production while demonstrating the value of combining classical microbiological techniques with modern bioinformatics for the characterization of plant growth-promoting bacteria

2. Materials and Methods

2.1 Study Design

The present study was designed as a comparative experimental investigation to evaluate the qualitative production of indole-3-acetic acid (IAA) by three bacterial species using broth- and agar-based Salkowski assays. Phenotypic observations were subsequently integrated with comparative genome-based bioinformatic analyses to investigate the genetic basis of auxin biosynthesis. The experimental workflow consisted of bacterial cultivation, qualitative IAA detection under tryptophan-supplemented conditions, photographic documentation of colour development, and computational analysis of auxin biosynthetic pathways using publicly available genomic resources.

2.2 Bacterial Cultures

Three bacterial species representing metabolically diverse plant growth-promoting bacteria were selected for comparative evaluation. Pure laboratory cultures of Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis maintained in the Department of Microbiology were used throughout the study. Before experimentation, the cultures were revived on Luria–Bertani (LB) agar and incubated at 37 ± 2°C for 24 h to obtain fresh, actively growing colonies. Single well-isolated colonies were subsequently used for all qualitative assays to ensure culture purity and reproducibility.

2.3 Culture Media and Reagents

Luria–Bertani (LB) broth and LB agar were employed for bacterial cultivation and qualitative screening. L-Tryptophan was supplemented to the medium at an appropriate concentration to induce auxin biosynthesis. Salkowski reagent was freshly prepared by mixing 1 mL of 0.5 M ferric chloride (FeCl₃) with 50 mL of 35% perchloric acid (HClO₄) and stored in an amber bottle until use. All media and glassware were sterilized by autoclaving at 121°C under 15 psi pressure for 15 min before use [32].

2.4 Qualitative Detection of Indole-3-Acetic Acid in Liquid Culture

A loopful of each bacterial culture was inoculated separately into sterile LB broth supplemented with L-tryptophan and incubated at 30°C under shaking conditions (80 rpm) for 72 h. Following incubation, an aliquot of each culture was mixed with freshly prepared Salkowski reagent in sterile glass test tubes. The reaction mixtures were incubated at room temperature in the dark for 30 min to allow colour development. Formation of yellow, orange, pink, or reddish coloration was considered indicative of the presence of indolic compounds, suggesting qualitative production of indole-3-acetic acid. All experiments were performed in triplicate to ensure reproducibility [33].

2.5 Qualitative Detection of Indole-3-Acetic Acid on Solid Medium

Fresh bacterial cultures were streaked individually onto LB agar plates supplemented with L-tryptophan and incubated at 30°C for 48 h. After visible colony development, sufficient freshly prepared Salkowski reagent was carefully added to the agar surface to completely cover the colonies. The plates were allowed to stand at room temperature for approximately 20 min to facilitate the colour reaction. Development of orange to pink coloration around bacterial colonies was interpreted as evidence of qualitative indole-3-acetic acid production. Representative plates were photographed under identical illumination conditions for comparative analysis [34].

2.6 Phenotypic Evaluation

The qualitative response of each bacterial species was assessed based on visible colour development in both liquid and solid media. To facilitate comparative interpretation, colour intensity was recorded using a visual scoring system adapted for qualitative screening (Table 1). Since the study focused on comparative phenotypic evaluation, no quantitative estimation of IAA concentration was performed.

2.7 Genome Retrieval and Identification of Auxin Biosynthetic Genes

Whole-genome sequences of Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis were retrieved from the National Center for Biotechnology Information (NCBI) Genome database. Reference genome assemblies with complete annotation were selected to ensure consistency during comparative analysis. Functional annotation files and corresponding protein sequences were downloaded for each organism.

Genes reported to participate in bacterial indole-3-acetic acid biosynthesis were identified through literature-guided searches and annotation mining. Particular emphasis was placed on genes involved in tryptophan-dependent pathways, including ipdC (indole-3-pyruvate decarboxylase), iaaM (tryptophan monooxygenase), iaaH (indoleacetamide hydrolase), trpA, trpB, trpC, trpD, trpE, and aldehyde dehydrogenase-encoding genes. The presence or absence of these genes in each bacterial genome was recorded and comparatively analyzed [35].

2.8 Comparative Analysis of Auxin Biosynthetic Genes

Identified auxin biosynthetic genes were compared among the three bacterial species to investigate similarities and differences in their genetic potential for indole-3-acetic acid production. Gene distribution, predicted protein length, functional annotation, and associated biosynthetic pathways were examined. Comparative visualization of gene presence and absence was performed using heat maps and schematic pathway diagrams to facilitate interpretation of species-specific differences.

2.9 Protein Sequence Retrieval and Physicochemical Characterization

Protein sequences encoded by selected auxin biosynthetic genes were retrieved from the UniProt and NCBI Protein databases. The physicochemical properties of the proteins, including amino acid length, molecular weight, theoretical isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity (GRAVY), were calculated using the ProtParam tool available through the ExPASy server. These analyses provided preliminary insights into the structural stability and biochemical characteristics of enzymes associated with auxin biosynthesis [36].

2.10 Conserved Domain and Functional Annotation

Functional domains within the selected proteins were identified using InterPro, Pfam, and the NCBI Conserved Domain Database (CDD). Conserved catalytic residues, functional motifs, and enzyme family classifications were examined to evaluate structural conservation among homologous proteins from the three bacterial species. The predicted domains were correlated with previously reported functions of enzymes involved in bacterial auxin biosynthesis [37].

2.11 Multiple Sequence Alignment

Multiple sequence alignment was performed using Clustal Omega to assess sequence conservation among homologous auxin biosynthetic proteins. Conserved catalytic regions and functional motifs were identified by integrating the alignment results with conserved-domain annotations from InterPro, Pfam, and the NCBI Conserved Domain Database (CDD) [39].

2.12 Phylogenetic Analysis

Comparative sequence analysis of representative auxin biosynthetic proteins was performed to assess evolutionary relatedness among the selected bacterial species. Multiple sequence alignments were generated using Clustal Omega to evaluate sequence conservation among homologous proteins from Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis. Conserved amino acid regions and sequence similarities were examined to assess functional conservation among the selected proteins. The observed similarities were interpreted in the context of conserved protein functions and bacterial phylogeny, providing insights into the evolutionary conservation of proteins involved in the tryptophan-dependent indole-3-acetic acid (IAA) biosynthetic pathway [40].

2.13 Three-Dimensional Protein Structure Prediction

Structural characteristics of representative auxin biosynthetic proteins were assessed using functional annotations available from UniProt, InterPro, Pfam, and the NCBI Conserved Domain Database (CDD). Conserved domains, predicted structural features, and enzyme family classifications were comparatively analysed to evaluate the structural conservation of homologous proteins from Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis. The observed structural similarities were interpreted in relation to their predicted roles in the tryptophan-dependent indole-3-acetic acid (IAA) biosynthetic pathway [41].

2.14 Molecular Docking Analysis

To investigate the interaction of L-tryptophan with auxin biosynthetic enzymes, molecular docking studies were performed using the CB-Dock2 online docking server, which integrates automatic cavity detection with the AutoDock Vina scoring algorithm. Three-dimensional protein structures of the selected enzymes were obtained from the AlphaFold Protein Structure Database, while the three-dimensional structure of L-tryptophan was downloaded from the PubChem database in SDF format. The protein structure and ligand were uploaded to the CB-Dock2 server without manual modification. The server automatically identified potential ligand-binding cavities, generated docking grids, and performed docking calculations using AutoDock Vina. For each protein, the cavity with the lowest (most negative) Vina binding score was selected as the most probable binding site. The docking results were evaluated based on binding affinity (kcal/mol) and the amino acid residues predicted to interact with the ligand within the selected binding pocket. Hydrogen bond analysis was not included because the CB-Dock2 output did not consistently identify hydrogen bonding interactions for all protein–ligand complexes. The predicted binding affinities and interacting residues were subsequently compared among the selected auxin biosynthetic enzymes to assess their potential involvement in L-tryptophan recognition [42].

2.15 Integration of Experimental and Bioinformatic Data

Phenotypic observations obtained from broth- and agar-based Salkowski assays were integrated with genomic and protein-level analyses to evaluate the relationship between observable auxin-producing characteristics and the predicted genetic potential of each bacterial species. Comparative interpretation was performed to determine whether qualitative differences in colour development corresponded with the presence of key auxin biosynthetic genes, conserved catalytic domains, and predicted substrate-binding characteristics of enzymes involved in indole-3-acetic acid biosynthesis.

Figure 4. Bar graph showing comparative qualitative scores obtained from broth- and agar-based assays.

The qualitative assessment of indole-3-acetic acid (IAA) production revealed notable differences among the three bacterial isolates. Bacillus subtilis exhibited the highest IAA-producing potential, demonstrating a very strong reaction in the broth assay and a strong reaction on agar, indicating its superior capacity for IAA synthesis under the tested conditions. In comparison, Serratia marcescens showed moderate IAA production in broth but only a weak reaction on agar, suggesting an intermediate ability to synthesize IAA that may be influenced by the growth medium. Pseudomonas aeruginosa displayed weak IAA production in the broth assay and a moderate reaction on agar, reflecting the lowest overall IAA-producing potential among the tested isolates despite its relatively improved performance on solid medium. Collectively, these findings indicate that Bacillus subtilis is the most promising IAA-producing isolate, whereas Serratia marcescens and Pseudomonas aeruginosa exhibited comparatively lower auxin-producing capacities, with medium-dependent variations in their qualitative responses.

 

 

3.4 Comparative Analysis of Auxin Biosynthetic Genes

Table 5. Comparative Analysis of Auxin Biosynthetic Genes  

Key note – ✓ = Annotated gene present in the reference genome.
✗ = No annotated ortholog detected in the reference genome.
✓* = An aldehyde dehydrogenase family gene is present, although it is not necessarily annotated with the exact gene symbol aldH.

3.5 Physicochemical Properties of Selected Auxin Biosynthetic Proteins

3.8 Phylogenetic Analysis

3.9 Structural Annotation of Representative Auxin Biosynthetic Proteins 3.10 Molecular Docking Analysis

4. Discussion

The present study integrated qualitative laboratory assays with comparative genome analysis, molecular docking, multiple sequence alignment, and phylogenetic analysis to evaluate the auxin biosynthetic potential of Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis. Combining experimental observations with bioinformatic analyses provided a comprehensive understanding of the molecular basis underlying bacterial indole-3-acetic acid (IAA) production.

Qualitative screening using Salkowski reagent demonstrated distinct differences in IAA production among the three bacterial species. Bacillus subtilis exhibited the strongest colour development in both broth and agar assays, indicating the highest auxin-producing potential. Serratia marcescens showed moderate activity, whereas Pseudomonas aeruginosa displayed comparatively weaker colour development. These observations suggest that bacterial species differ considerably in their ability to synthesize IAA despite possessing similar precursor biosynthetic pathways.

Comparative genome analysis revealed that all three organisms possess the complete set of core tryptophan biosynthetic genes (trpA, trpB, trpC, trpD, and trpE), confirming their genetic capacity to synthesize L-tryptophan, the principal precursor for IAA biosynthesis. However, the commonly reported auxin pathway genes ipdC, iaaM, and iaaH were not detected in the selected reference genomes. This finding suggests that these bacteria may utilize alternative tryptophan-dependent pathways or functionally equivalent enzymes that remain unannotated in current genome databases.

To investigate the structural basis of substrate recognition, representative aldehyde dehydrogenase proteins from each organism were selected based on experimentally validated UniProt annotations. AlphaFold-predicted protein structures were analysed using CB-Dock2 with L-tryptophan as the ligand. All proteins exhibited favourable binding pockets and negative docking scores, indicating the potential for stable substrate recognition. Although hydrogen-bond interactions were not consistently observed, the predicted binding poses suggest that hydrophobic and van der Waals interactions contribute substantially to ligand stabilization within the active site. These findings provide computational support for the possible involvement of aldehyde dehydrogenases in the terminal oxidation step of tryptophan-dependent auxin biosynthesis.

Multiple sequence alignment demonstrated high conservation of catalytically important regions among TrpA, TrpB, TrpC, TrpD, TrpE, and aldehyde dehydrogenase proteins. Conserved functional motifs identified in these enzymes indicate preservation of catalytic activity despite evolutionary divergence. In particular, the conserved catalytic cysteine and glutamate residues characteristic of aldehyde dehydrogenases suggest maintenance of their enzymatic function across the investigated bacterial species.

Phylogenetic analysis further supported these observations by showing that the tryptophan biosynthetic proteins of Pseudomonas aeruginosa and Serratia marcescens clustered closely together, reflecting their common Gram-negative evolutionary origin. In contrast, Bacillus subtilis consistently formed a separate branch, consistent with its Gram-positive lineage. Aldehyde dehydrogenases exhibited relatively greater sequence divergence than the tryptophan biosynthetic enzymes, reflecting the broader functional diversity of this enzyme family while retaining conserved catalytic domains.

The integrated experimental and computational results indicate that variations in IAA production cannot be explained solely by the presence of auxin biosynthetic genes. Instead, differences in gene regulation, enzyme activity, metabolic efficiency, and cellular physiology are likely to influence the observed phenotypic differences. The superior qualitative performance of Bacillus subtilis may therefore result from more efficient metabolic regulation rather than differences in gene content alone.

A major strength of this study is the integration of wet-laboratory observations with comparative genomics and structural bioinformatics, allowing experimental findings to be interpreted within a molecular framework. However, the study was limited to qualitative colour-based assays, and the docking analyses represent computational predictions that require experimental validation. Future studies employing quantitative IAA estimation, gene expression analysis, enzyme characterization, and functional validation will provide a more comprehensive understanding of bacterial auxin biosynthesis.

The present findings demonstrate that comparative genomics, protein structural analysis, and molecular docking effectively complement conventional laboratory screening for plant growth-promoting bacteria. This integrated workflow provides a valuable framework for identifying bacterial strains with auxin biosynthetic potential and contributes to a better understanding of the molecular mechanisms underlying microbial phytohormone production.

5. Conclusion

The present study successfully integrated qualitative experimental screening with comparative genomic and structural bioinformatic analyses to evaluate the auxin biosynthetic potential of Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis. Qualitative detection using Salkowski reagent demonstrated clear differences in IAA production among the tested organisms, with Bacillus subtilis exhibiting the strongest response, followed by Serratia marcescens, whereas Pseudomonas aeruginosa displayed comparatively weaker production.

Comparative genome analysis confirmed that all three bacterial species possess the complete core tryptophan biosynthetic pathway (trpA–trpE), supporting their capacity to synthesize the precursor required for auxin production. Although canonical auxin pathway genes (ipdC, iaaM, and iaaH) were absent from the reference genomes, the presence of conserved aldehyde dehydrogenase family proteins suggests that alternative tryptophan-dependent pathways may contribute to IAA biosynthesis. Molecular docking further demonstrated favourable interactions between L-tryptophan and representative aldehyde dehydrogenases, while multiple sequence alignment and phylogenetic analyses confirmed strong conservation of catalytically important residues and evolutionary relationships among the selected proteins.

Collectively, the findings indicate that auxin production depends not only on the presence of biosynthetic genes but also on enzyme functionality and cellular regulation. The integrated experimental–bioinformatic workflow established in this study provides a robust strategy for evaluating plant growth-promoting bacteria and may facilitate the identification of promising microbial candidates for sustainable agricultural applications.

6. Future Prospects

The present investigation provides a foundation for further exploration of bacterial auxin biosynthesis through advanced molecular and biochemical approaches. Future studies should focus on quantitative estimation of IAA using high-performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS), or gas chromatography–mass spectrometry (GC–MS) to validate the qualitative observations obtained in this study.

Expression analysis of key tryptophan biosynthetic and aldehyde dehydrogenase genes using quantitative real-time PCR (qRT-PCR) or transcriptome sequencing would provide insights into regulatory mechanisms governing auxin biosynthesis under different environmental conditions. Gene knockout or overexpression studies could further establish the precise contribution of individual enzymes to IAA production.

Detailed enzyme characterization, including purification, kinetic analysis, substrate specificity, and crystallographic studies, would improve understanding of catalytic mechanisms involved in bacterial auxin biosynthesis. Molecular dynamics simulations and free-energy calculations may also complement molecular docking by providing information on protein flexibility and long-term ligand stability.

Evaluation of the selected bacterial strains under greenhouse and field conditions will be necessary to determine their effectiveness as plant growth-promoting rhizobacteria. Studies investigating seed germination, root architecture, nutrient uptake, stress tolerance, and crop productivity will provide practical evidence supporting their agricultural applications. Furthermore, interactions between bacterial auxin production and other plant growth-promoting traits such as phosphate solubilization, siderophore production, nitrogen fixation, ACC deaminase activity, and biofilm formation should be investigated to identify multifunctional microbial inoculants.

The integration of comparative genomics, structural bioinformatics, functional genomics, metabolomics, and experimental microbiology represents a promising strategy for accelerating the discovery of efficient plant growth-promoting bacteria suitable for environmentally sustainable agriculture.

7. Study Limitations

The present study has several limitations that should be considered while interpreting the findings. Indole-3-acetic acid (IAA) production was evaluated using qualitative Salkowski colour reactions, which provide only a preliminary assessment of auxin production and do not quantify IAA concentrations. The molecular docking analysis performed using CB-Dock2 predicts potential protein–ligand interactions based on structural models but does not directly demonstrate enzymatic activity or substrate specificity under biological conditions. Furthermore, although comparative genome analysis confirmed the presence of genes associated with tryptophan biosynthesis, the presence of these genes does not necessarily indicate their expression or functional activity under the experimental conditions employed. The investigation was also limited to three representative bacterial species, which may not fully represent the diversity of auxin-producing microorganisms. In addition, gene expression analysis, enzyme kinetics, metabolomic profiling, and plant inoculation studies were beyond the scope of the present work. Despite these limitations, the integration of qualitative experimental observations with comparative genomics, structural bioinformatics, molecular docking, multiple sequence alignment, and phylogenetic analysis provides valuable preliminary evidence supporting the auxin biosynthetic potential of the investigated bacterial species and establishes a strong foundation for future functional and quantitative investigations.

8. Ethical Considerations for the Use of Artificial Intelligence (AI) in Academic Writing

Artificial intelligence (AI)-assisted writing tools were used solely to support language refinement during the preparation of this manuscript. These tools were employed to improve grammar, spelling, punctuation, sentence structure, and overall readability, and were not used to generate research questions, develop the study design, analyze data, interpret results, or formulate the scientific conclusions. All intellectual contributions, including the conceptualization of the research, methodology, data collection, analysis, interpretation, and final manuscript preparation, were undertaken exclusively by the authors. Every AI-generated suggestion was carefully reviewed, verified, and modified where necessary to ensure the accuracy, originality, and integrity of the scientific content. The authors accept full responsibility for the content of the manuscript and affirm that the use of AI-assisted writing tools complied with the ethical standards, institutional policies, and the AI-use guidelines of the target journal.

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