# Identification of overlooked tryptophan catabolism pathways in bacteria: a focus on rhizobacteria

Adrian Lautrey, Laurent Legendre, Florence Wisniewski-Dyé, Claire Prigent-Combaret

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Adrian Lautrey, Laurent Legendre, Florence Wisniewski-Dyé, Claire Prigent-Combaret. Identification of overlooked tryptophan catabolism pathways in bacteria: a focus on rhizobacteria. Heliyon, 2025, 11 (15), pp.e44009. 10.1016/j.heliyon.2025.e44009. hal-05376514

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# Identification of overlooked tryptophan catabolism pathways in bacteria: a focus on rhizobacteria

Adrian Lautrey, Laurent Legendre, Florence Wisniewski-Dyé, Claire Prigent-Combaret

Université de Lyon, Ecologie Microbienne, CNRS UMR-5557, INRA UMR-1418, Université Lyon1, VetAgroSup, 43 boulevard du 11 novembre 1918, Villeurbanne, 69622, France

## ARTICLE INFO

**Keywords:**
*   Tryptophan
*   Anthranilate
*   Kynurenine
*   *Azospirillum*
*   Genome analysis

## ABSTRACT

Tryptophan (Trp) is primarily catabolized via the kynurenine or indolic pathways in bacteria, resulting in the production of biologically active compounds that are crucial for bacteria-host interactions. While Trp-catabolism has been extensively studied in gut bacteria, research on bacteria associated with plant roots has primarily focused on Trp-derived auxins. Here, a broader *in silico* analysis of Trp-catabolic pathways across the bacterial kingdom was performed. A broad distribution of Trp-degradation pathways was highlighted. First, the kynurenine pathway genes (leading to quinolinate) were more broadly found than key genes of other Trp-degradative pathways. Specifically, the anthranilate pathway that constitutes the initial steps of the kynurenine pathway, was predominantly found in soil-borne or plant-associated bacteria. Second, an evolutionary analysis of genes from the kynurenine pathway suggested a vertical transmission of the tryptophan-2,3-dioxygenase-encoding gene but a likely horizontal transmission of genes encoding kynurenine formamidase isoenzymes. In contrast, the kynureninase exhibited an unconventional evolutionary pattern, differentiating strains possessing the anthranilate pathway from those possessing the kynurenine pathway. Third, a focus on the plant growth-promoting *Azospirillum* genus revealed that it harbours two non-co-occurring Trp-catabolic pathways with putative distinct impacts on plants, the auxin-producing pathway in the *brasilense* clade and the anthranilate pathway in the *lipoferum* clade.

## 1. Introduction

The aromatic amino acid tryptophan (Trp), the least abundant amino acid in the kingdom of life, holds significant importance as an essential amino acid for all living organisms. In most bacteria, Trp is primarily synthesized de novo via the shikimate pathway or from anthranilate [1]. However, Trp can also be acquired from external sources, such as through protein degradation, organic matter, or absorption of both free and complexed forms. Beyond its primary role in protein synthesis, Trp plays versatile functions through various catabolic pathways. Among bacteria, two major Trp catabolic pathways are recognized: the kynurenine pathway and the pathway involving indolic compounds. These pathways lead to the synthesis of numerous bioactive compounds, often playing a critical role in mediating interactions between bacteria and their eukaryotic hosts.

<footnote>* Corresponding author. UMR CNRS 5557 Ecologie Microbienne, Université Claude Bernard Lyon 1, 43 bd du 11 novembre 1918, Villeurbanne, F-69622, France.</footnote>

<footnote>E-mail address: claire.prigent-combaret@univ-lyon1.fr (C. Prigent-Combaret).</footnote>
Recent interest in bacterial Trp catabolism has provided insights into the interactions between gut bacteria and their animal hosts. The kynurenine pathway has been identified in bacteria and animals, but is absent in plants. This pathway plays a pivotal role by modulating free Trp levels in the host, with downstream effects on host metabolism [2]. This pathway generates several bioactive metabolites, including kynurenic acid, L-kynurenine, and anthranilate, which influence neuronal, metabolic, and protective functions (such as reactive oxygen species scavenging) in mammals. For instance, *Pseudomonas aeruginosa* PAO1 utilizes this pathway to trick the immune system during the infection process [3].

The pathways of Trp-derived indole compounds have been found mainly in bacteria and plants. Some compounds of this family have been shown to accelerate the cell cycle in eukaryotes (indole-3-acetic acid (IAA), [4]), modulate cell development (IAA, [4,5]) and affect the central nervous system (IAA, [6]; indole-3-propionic acid, [7]; indole, [8]). Trp-derived indolic compounds also act as modulators of the immune system and mucosal inflammation (IAA and indole carboxylic acid, [9,10]) by promoting mucus secretion and limiting intestinal inflammation (indole propionic acid, [11]). They also exhibit neuroprotective properties (indole propionic acid [12], and have been implicated in the regulation of incretin and insulin secretion [13]). Furthermore, Trp-derived indolic compounds display antifungal and antibacterial activity (indole propionic acid and indole [14–16]), and facilitate intricate molecular communication within microbial communities (indole and IAA, [17–19]).

The distribution of the Trp degradation pathway has been previously explored in gut bacteria, providing a comprehensive catalogue of these pathways in various gut-associated bacterial species [12]. These analyses revealed an enrichment of Trp metabolism pathways in *Actinobacteria*, *Firmicutes*, *Bacteroidetes*, *Proteobacteria*, and *Fusobacteria*, suggesting function-driven positive selection pressure. Despite many commonalities between animal intestines and plant roots ([20] for review), studies of Trp catabolism pathways in plant-associated bacteria (i.e., rhizobacteria) remain very limited to date.

Free Trp is released into the rhizosphere as part of a broader phenomenon involving the release of amino acids and small molecules: the rhizodeposition. These organic remnants, termed rhizodeposits, originate from various sources including detached root cells (particularly root cap cells), lysates, mucilages, and exudates [21,22]. The concentration of Trp ranges from 2.8 to 5.3 ng per 4-day-old tomato (Solanaceae) seedling per day and from 290 to 390 ng per radish (Brassicaceae) root per day [23,24]. Current research on Trp

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Trp degradation pathways in bacteria.</figcaption></figure>

The kynurenine pathway (solid line) facilitates the conversion of tryptophan (Trp) into quinolinate. A sub-branch of this pathway, the anthranilate pathway (dotted line), leads to the production of anthranilate from Trp and involves the first three enzymatic steps of the kynurenine pathway. The key enzymes of the kynurenine pathway include: tryptophan-2,3-dioxygenase (TDO) (Blue); kynurenine formamidase (KFA/AFMID) (Green/Yellow); kynureninase (KYNU) (Red); kynurenine transaminase (KAT) (Orange); kynurenine monooxygenase (KMO) (Plum); and 3-hydroxyanthranilate 3,4-dioxygenase (HAD) (Dark grey). Alternatively, quinolinate can be synthesized via the aspartate-to-quinolinate pathway. Additionally, several key enzymes have been identified as playing critical roles in other Trp degradation pathways.

The biosynthetic pathways of indolic compounds production can be broadly classified into four routes: the indole-3-pyruvate (IPyA) pathway, the indole-3-acetamide (IAM) pathway, the indole pathway, and the tryptamine pathway. The IPyA, IAM, and tryptamine pathways lead to the synthesis of auxins, while the indole pathway results in the production of indole. The key enzymes for each pathway are: indole pyruvate decarboxylase/phenylpyruvate decarboxylase (IpdC/PpdC) (Purple) for the IPyA pathway; tryptophan-2-monooxygenase (IaaM) (Pink) for the IAM pathway; tryptophanase (TnaA) (Light pink) for the indole pathway; and tryptophan decarboxylase (TDC) or aromatic amino acid decarboxylase (Brown) for the tryptamine pathway.
catabolism in plant-rhizobacteria interactions primarily focuses on the synthesis of auxin-type plant hormones, particularly indole-3-acetic acid (IAA). Auxin synthesis has been identified as a key feature in many rhizobacteria such as *Azospirillum baldaniorum* Sp245 which significantly promotes plant growth by enhancing cell division and elongation [25]. However, little is known on Trp degradation in non-auxin producing strains, as the plant root associated strain *Azospirillum* sp. 4B (formerly *A. lipoferum* 4B) [26]. In general, the focus on the importance of indolic phytohormones over the last fifty years has left other Trp degradation pathways largely unexplored. Notably, there is limited knowledge about alternative Trp degradation pathways in strains interacting with plants and the potential impacts of their metabolic by-products on plant development. Some metabolites, such as L-kynurenine and serotonin, have been shown to affect plant growth; for example, L-kynurenine inhibits auxin synthesis, while serotonin has demonstrated multiple beneficial effects on plant growth [27–31].

Therefore, a comprehensive analysis of Trp catabolism genes in plant-associated bacteria is likely to enhance our understanding of these pathways' roles in plant-bacteria interactions.

This study aims to characterize in silico Trp degradation pathways across the bacterial kingdom, including a representative panel of rhizobacteria along with *Azospirillum* sp. 4B. The widespread presence of kynurenine pathway genes in rhizobacteria prompted a detailed analysis of each biosynthetic enzymatic step. Given the source of isolation of strains from the *Azospirillum* genus, this study highlights the potentially adaptive, though non-essential, role of these Trp degradation pathways in plant-bacteria interactions. The identification of gene homologs associated with the anthranilate pathway in the model strain *Azospirillum* sp. 4B provides a foundation for future functional studies of this pathway in growth-promoting rhizobacteria and its potential role in mediating plant-bacteria interactions.

## 2. Methods

### 2.1. Selection of the Trp-degradative pathways

Metabolic pathways, metabolites, and enzymes were sourced from the KEGG database [32]. The occurrence of the pathways was predicted based on homologous genes and proteins found in annotated bacterial genomes. The bacterial Trp degradation pathways are outlined below and depicted schematically in Fig. 1. Supplementary Table S1 provides a summary of the seed sequence information for the enzymes involved in these pathways.

#### 2.1.1. Anthranilate pathway (tryptophan to anthranilate pathway)

The anthranilate pathway encompasses the three initial steps of the kynurenine pathway. The anthranilate pathway involves the tryptophan-2,3-dioxygenase encoded by *kynA* (TDO, EC 1.13.11.11), the kynurenine formamidase encoded by *kynB* (KFA, EC 3.5.1.9) and the kynureninase encoded by *kynU* (KYNU EC 3.7.1.3) along with the kynurenine transaminase encoded by the putative *kynT* (KAT, EC 2.6.1.7). These enzymes sequentially convert Trp into *N*-formyl-L-kynurenine (NFK), L-kynurenine (LK) and anthranilic acid (AA). In addition to this primary pathway, kynurenine acid (KA) is a secondary product derived from LK (Fig. 1). However, the enzyme that catalyses this reaction, kynurenine aminotransferase (KAT), has not been formally identified in bacteria, although KAT activity appears to be widespread. Indeed, KA formation from LK can also be facilitated by aspartate aminotransferase (AspAT), which shares sequence and structural similarities with KAT [33–36]. The *kynB* gene designation incorrectly groups two evolutionary independent genes encoding two structurally different kynurenine formamidase izoenzymes in bacteria: (i) the kynurenine formamidase with cyclase-like activity (metal-based hydrolase catalysis - KFA) has been extensively and solely characterized in bacteria [37,38] while (ii) the kynurenine formamidase exhibiting hydrolase activity (commonly referred as esterase/lipase and alpha/beta hydrolase - AFMID) has been found mainly in animals, fungi and in some bacteria, such as *Streptomyces coelicolor* [39]. The gene encoding KFA is proposed to be named *kynB*, and the gene encoding AFMID is proposed to be named *afmid*.

#### 2.1.2. Kynurenine pathway (tryptophan to quinolinate pathway)

The kynurenine pathway encompasses the anthranilate pathway along with two additional enzymatic steps. These steps involve kynurenine monooxygenase encoded by *kmo* (KMO, EC 1.14.13.9) and 3-hydroxyanthranilate 3,4-dioxygenase encoded by *nbaC* (HAD, EC 1.13.11.6). Sequential action of these enzymes results in the production of 3-OH-L-kynurenine, 3-OH-anthranilic acid (3-OH-AA) and finally quinolinate. Additionally, xanthurenic acid can be produced as by-products through the action of KAT on 3-hydroxy-L-kynurenine.

#### 2.1.3. Acid indole-3-pyruvate pathway

The acid indole-3-pyruvate (IPyA) pathway is considered as the main route for auxin biosynthesis in plants and many plant-beneficial rhizobacteria [40–43]. The central and specific enzyme of the IPyA pathway is the indole pyruvate decarboxylase/phenylpyruvate decarboxylase (IpdC/PpdC encoded by the *ipdC/ppdC* gene)(Fig. 1). Inoculation of *Arabidopsis thaliana* with *A. baldaniorum* Sp245 mutated in this gene (strain FAJ0009) yielded a phenotype similar to the non-inoculated control. This result underscores the critical role of this enzyme in the IPyA pathway and the interaction between *A. baldaniorum* Sp245 and *A. thaliana* [44].

#### 2.1.4. Indole-3-acetamide pathway

The indole-3-acetamide (IAM) pathway is particularly prevalent in tumor-forming phytopathogenic bacteria. The IAM pathway involves the key enzyme tryptophan-2-monooxygenase (IaaM-EC 1.13.12.3- encoded by *iaaM/tmsI*) (Fig. 1) [45]. Inactivation of the
IAM pathway leads to a near-complete cessation of tumor formation [46].

### 2.1.5. Indole pathway

The indole pathway is mediated by tryptophanase (TnaA -EC 4.1.99.1- encoded by tnaA) and facilitates the conversion of Trp to indole (Fig. 1).

### 2.1.6. Tryptamine pathway

The tryptamine pathway is driven by the key enzyme tryptophan decarboxylase or a low-specific aromatic amino acid decarboxylase (EC 4.1.1.28–4.1.1.105) (Fig. 1).

Aside from these pathways, the serotonin pathway and the tryptophan side chain oxydase pathway remain speculative in bacterial systems, despite some metabolic evidence. Consequently, these pathways are not addressed in the current study.

## 2.2. Identification of the Trp degradative pathways

Homologous sequences to the seed sequences were identified using the BLASTP algorithm against a dataset comprising 131 complete genomes from the MicroScope-Genoscope and NCBI databases (as of October 1, 2023) (Supplementary Table S2). This dataset included 26 *Azospirillum* genomes and 105 genomes representing a diverse array of bacterial species, encompassing 37 strains isolated from soil or plant environments. [47]. A BLASTP search was conducted using amino acid seed sequences with known functional assignments to identify homologs within bacterial genomes. The inclusion thresholds for BLASTP were set to a p-value <10<sup>-40</sup>, a query coverage >80 % and a percentage similarity >20 %. In the case of homologs searches based on eukaryotic sequences, the inclusion thresholds for BLASTP were decreased and set to a p-value <10<sup>-05</sup> and a query cover >60 %, with no similarity threshold. This preliminary selection was further refined based on additional criteria, as detailed below and summarized in Supplementary Fig. S1.

## 2.3. Cleaning of the protein sequences of the anthranilate pathway

Given the limitations of the BLASTP approach for enzymes containing generic domains, such as hydrolases, the current study employed enzyme homology analysis in conjunction with genomic context (where necessary) and the conservation of critical residues to accurately predict the pathway. Specifically, the genomic context of *kynB/afmid* genes was analysed, and only sequences located within ±5 kbp of the *kynA* or *kynU* loci were retained. Protein sequences for the anthranilate pathway were then aligned using the Muscle algorithm within the Mega-X software. Key catalytic residues were compared to seed sequences, and only those sequences with strictly identical residues at these critical positions were conserved.

For TDO, the key residues retained were Y-24, F-51, E-61, R-117, H-240, and T-254 [48,49]. Residues at H-55-T and Y-113-F were excluded from consideration due to the absence of conservation at these positions in functionally characterized TDO from *Streptomyces coelicolor* [39]. For KYNU, key residues retained were T-96, S-98, F-129, D*-132, D*-201, H-204, YK-226-227, W-256, and T-282 (*amino acids subjected to targeted mutagenesis*) [50]. For kynurenine formamidase with cyclase-like activity (KFA), important retained residues were H-50, H-54, D-56, D-60, H-161, and E-173 [38,51]. Residues at position W-20, corresponding to a substrate-binding site, were not considered in the data curation. For kynurenine formamidase with a hydrolase activity (AFMID), key retained residues were S-157, D-244, H-276 as well as the HGGXW motif at position 86–90 [52–54]. For all key residues, identical amino acids or substitutions with similar properties were accepted. The Snapgene Viewer (V5.2.3) was utilized for the graphical representation of multiple alignments (SnapGene software - wwwControlledVBox.com).

## 2.4. Tree inference

Previously aligned sequences were utilized and regions exhibiting ambiguity, high variability, or gaps being removed from further analysis using the Gblocks program (version 0.91.1) available from NGPhylogeny.fr [55,56]. A less stringent filtering criterion was applied to homologs of KFA from *Eukaryote* to achieve a minimum of 100 residues for tree construction. To infer a taxonomic tree, a multilocus sequence analysis (MLSA) was conducted using three taxonomic markers: RpoD, RecA, and GyrA. The corresponding protein sequences were concatenated prior to alignment.

Distance-based phylogenetic trees were generated through the Neighbor-Joining (NJ) algorithm (NJ program). Tree topology was assessed via bootstrap analysis with 1000 replicates using MEGA-X (version 10.2.2). Maximum Likelihood (ML) trees were constructed with Mega X (version 10.2.2) employing the Poisson substitution model. Where feasible, trees were rooted using the *Bacilli* phylum. Bootstrap analyses with 100 replicates were performed, generating bootstrap values up to 70 on the best-scoring ML tree. Given that ML and distance-based trees corroborated each other, only the ML trees are presented in this study. The inferred trees were imported into iTOL v6.5.8 for the integration of protein distribution datasets (iTOL, https://itol.embl.de).

# 3. Results and discussion

## 3.1. The proteins of the kynurenine pathway are more widely distributed in the bacterial kingdom than proteins of other Trp degradative pathways

Tryptophan metabolism in bacteria is characterized by two principal pathways: the kynurenine pathway and the pathways
involving indolic compounds. The kynurenine pathway is primarily mediated by tryptophan dioxygenase (TDO) and kynureninase. The indolic pathways are divided into four distinct pathways: (i) the indole pyruvic acid pathway, which involves indole pyruvate/phenyl pyruvate decarboxylase (IpdC/PpdC); (ii) the indole acetamide pathway, involving tryptophan monooxygenase (IaaM); (iii) the indole pathway, requiring tryptophanase (TnaA); and (iv) the tryptamine pathway, mediated by tryptophan decarboxylase, also known as aromatic amino acid decarboxylase.

A search for these proteins was carried out using BLASTP on a dataset of 131 genomes representative of the bacterial kingdom. A Maximum Likelihood phylogenetic tree, based on the taxonomic markers RpoD, RecA, and GyrA, was constructed for 107 selected bacterial genomes (Fig. 2). The clustering and clade positioning were generally in agreement with the established taxonomic tree of life [47].

The distribution analysis of Trp degradation pathways revealed that genes associated with the kynurenine pathway were more widely distributed across the bacterial kingdom compared to those of other Trp degradation pathways, although their distribution was discontinuous, as previously noted [57]. Certain bacterial classes, such as *Chlorobiia*, *Spirochaetia*, *Brocadia*, *Chlamydiia*, *Fusobacteriia*, *Thermotogae*, *Aquificae*, and *Cyanophyceae*, were found to lack all kynurenine pathway proteins (Fig. 2). Conversely, 48 strains (45%) were identified as possessing at least one protein involved in the kynurenine pathway, spanning various phyla such as *Proteobacteria* (now *Pseudomonadota*), *Deinococcota*, *Bacillota* and *Actinomycetota*. Noteworthy, bacterial taxa possessing these genes appeared to be over-represented in plant or soil-associated strains such as *Xanthomonas*, *Pseudomonas*, *Streptomyces* and particularly in *Bradyrhizobium*, *Ensifer*, *Mezorhizobium* and *Azospirillum* sp. 4B which are all known to interact with plants and promote their growth (Plant Growth-Promoting Rhizobacteria (PGPR)) [58–61]. In fact, 27 out of 37 strains (73%) isolated from soil or plant environments possessed genes potentially encoding TDO and/or kynureninase. The presence of the kynurenine pathway in beneficial rhizobacterial

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Distribution of the kynurenine, auxin, indole and tryptamine pathways in a representative subset of bacterial species.</figcaption></figure>

On a taxonomic Maximum Likelihood tree inferred using the RpoD, RecA, and GyrA taxonomic markers, the distribution of the kynurenine (blue circles), auxins (purple and pink circles), indole (light pink circles) and tryptamine (brown circles) pathways was based on the identification of the key enzymes of these pathways. Green squares indicate strains isolated from soil or plants.
strains has not been previously documented.

In comparison, the distribution of indole pyruvate/phenyl pyruvate decarboxylase genes appeared sporadic across the bacterial
kingdom, consistent with earlier studies [62] (Fig. 2). Notably, the *ipdC/ppdC* gene was found in genomes of *Bacillus cereus*, *Bacillus
anthracis*, *Salmonella enterica*, *Pantoea ananatis*, *Rhodospirillum rubrum*, *Azospirillum baldaniorum* Sp245 as well as in six other strains.
The key enzyme of the IAM pathway, tryptophan-2-monooxygenase (IaaM), was identified in seven strains in our dataset (Fig. 2),
including strains of the genus *Streptomyces* and in *Pseudomonas fluorescens*. IaaM was also detected in a *Frankia discariae*, a
nitrogen-fixing symbiotic bacterium isolated from the root nodule of *Discaria trinervis*, and in *Agrobacterium tumefaciens*, a
well-documented phytopathogen causing crown gall [63] (Fig. 2). Tryptophanase (TnaA) was found in nine strains, particularly within
*Enterobacteria* and within the *Fusobacteria/Elusimicrobia* clade (Fig. 2). Surprisingly, TnaA was observed never to co-occur with key
enzymes of the kynurenine pathway in our dataset. Additionally, the enzyme involved in the tryptamine pathway (i.e. aromatic amino
acid decarboxylase) was found in a broad spectrum of strains, including *Actinobacteria* strains, some *Gamma-proteobacteria* strains and
occasionally in *Alpha-proteobacteria* (and a few other strains) but has never been found in *Beta-proteobacteria*. This decarboxylase is
proposed to have a multifaceted role in metabolizing aromatic amino acids, including Trp [64].

Finally, 38 strains, predominantly isolated from animals or aquatic environments, were found to lack any of the identified Trp
degradation enzymes. Taken together, the observed discontinuous distribution of Trp degradation pathways suggests multiple inde-
pendent events of gene loss or acquisition throughout evolutionary history. Overall, kynurenine pathway proteins were found to be
more widely distributed across the bacterial kingdom compared to key proteins of other Trp degradation pathways. Given their over-
representation in rhizobacteria, further focus is warranted on the distribution, conservation, and evolution of the kynurenine pathway,
particularly the core anthranilate pathway.

<figure><img src="image_5.png" /><figcaption>Fig. 3. Distribution of anthranilate and kynurenine pathway enzymes in a subset of strains representing the bacterial kingdom.</figcaption></figure>

The taxonomic tree was inferred using RpoD-RecA-GyrA concatenated amino acid sequences. Protein identification was done following the selection method described in Material and methods and in Supplementary Fig. S1. Indoleamine 2,3-dioxygenase (IDO) that displays an activity similar to that of TDO [65] was only found in *Gemmatiomonas aurantica*.
## 3.2. The anthranilate pathway exhibits greater prevalence compared to the kynurenine pathway in the bacterial kingdom

As mentioned above, the kynurenine pathway encompasses the entire process from Trp to quinolinate, involving several enzymes: tryptophan dioxygenase (TDO), kynurenine formamidase (KFA), kynureninase (KYNU), kynurenine monooxygenase (KMO), and 3-hydroxyanthranilate 3,4-dioxygenase (HAD). However, the initial three steps of this pathway are part of the anthranilate pathway, also referred to as the Trp to anthranilate pathway (Fig. 1). This pathway acts as a precursor for the synthesis of compounds beyond quinolinate. Consequently, we investigated the evolutionary trajectories of the two segments of the quinolinate pathway separately (Fig. 3).

Homologs of the three genes associated with the anthranilate pathway were identified in 28 strains (26 %) across various bacterial classes, including *Alpha-proteobacteria* (e.g., Rhodospirillales, *Rhodobacterales*, *Rhizobiales*), *Beta-proteobacteria*, *Gamma-proteobacteria*, *Deinococci*, *Bacilli*, and *Actinomycetes* (Fig. 3). Notably, two structurally distinct kynurenine formamidase families can participate in the anthranilate/kynurenine pathway (i.e KFA and AFMID). The distribution of these enzymes was non-uniform and did not cluster according to taxonomy. AFMID was observed in *Alpha-proteobacteria* and *Actinomycetes*, whereas KFA was predominantly found in *Beta-proteobacteria*, *Gamma-proteobacteria*, and *Bacilli*. Some deviations from this distribution (e.g., *Alcaligenes faecalis* and *Nitrospirillum amazonense*) suggest potential gene transfer events between bacterial classes (Fig. 3).

The absence of co-occurrence between these enzyme families indicates that kynurenine formamidase genes from one family may have been acquired via lateral gene transfer (LGT) and replaced those of the other family due to functional redundancy. An additional twenty strains exhibited an incomplete anthranilate pathway, containing at least one gene from the *kynA*, *kynB/afmid*, and *kynU* triad (*kyn* genes). The absence of *kynB/afmid* in certain strains may result from the stringency of homolog searches, suggesting that strains harboring *kynA* and *kynU* could potentially possess a complete anthranilate pathway.

The kynurenine pathway was found to be complete solely in *Myxococcus xanthus* (Fig. 3). The post-anthranilate enzymes of the kynurenine pathway (KMO and HAD) were detected in six other strains, including those from *Xanthomonadales*, *Flavobacteriia*, and one *Actinobacteria*-related strain. Overall, KMO was infrequently observed across the bacterial kingdom (8 %) and was consistently associated with KYNU (Fig. 3). The 3-hydroxyanthranilate 3,4-dioxygenase (HAD), encoded by the *nbaC* gene, was frequently found in association with KMO (64 %) and always with KYNU.

The two-step aspartate pathway, an alternative to the kynurenine pathway involving NadX/NadB and NadA (Fig. 1), was present in 76 % of the representative bacterial subset. Its distribution did not correlate with that of the anthranilate pathway (data not shown).

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Organization of genes of the anthranilate and kynurenine pathways.</figcaption></figure>

The taxonomic tree was inferred using RpoD-RecA-GyrA concatenated amino acid sequences. Next to the tree, organisation of the *kyn* genes (*kynA*, *kynB*, *afmid*, *kynU*, *kmo* and *nbaC*) and of genes of the other auxin pathways (*ipdC/ppdc*, *iaaM/tms1*) is depicted. *tnaA* was never found in association with the *kyn* genes among the selected dataset. The gene encoding tryptophan decarboxylase involved in the tryptamine pathway was not indicated. Only bacteria that possess at least two genes of the anthranilate pathway are presented.
However, Xanthomonadales and Flavobacteriales, which harbour kynurenine pathway-specific genes (KMO & HAD), lack the aspartate pathway, a finding consistent with previous studies [57].

In summary, the complete anthranilate pathway is more prevalent in the bacterial kingdom compared to the complete kynurenine pathway.

### 3.3. The anthranilate pathway genes are often organized in an operon-like structure

The organization of genes involved in the anthranilate pathway has previously been investigated in only a limited number of strains [37,39,57]. To expand upon these findings, we analysed the genetic organization of these genes in a broader selection of bacterial strains, specifically those possessing at least two kynurenine pathway-related genes (Fig. 4).

In strains with a complete anthranilate pathway, the three *kyn* genes were frequently found in close vicinity (20 out of 28 strains), and were sometimes organized in an operon-like structure (15 out of 20 strains), as observed in *Ralstonia picketti* or in the *Bacilli* clade (Fig. 4). The organization of the *kyn* locus even appeared highly conserved in *Beta-proteobacteria* compared to other taxa. Furthermore, *kyn* genes organisation exhibited significant variability due to multiple genome rearrangements, with heterogeneous gene organization observed among strains within the same genus (e.g., *Pseudomonas*, *Bradyrhizobium*, *Streptomyces*) (Fig. 4). In *Xanthomonadales* and *Myxococcus xanthus*, the *kynU*, *kmo*, and *nbaC* genes were co-localized and arranged similarly: *nbaC*, followed by a gene unrelated to the studied pathway, followed by *kynU* and *kmo* (Fig. 4).

Overall, the *kyn* genes often co-localize and are occasionally organized in an operon-like structure.

### 3.4. TDO and KFA sequences are highly conserved within the bacterial kingdom, unlike AFMID

Previous studies suggested that key genes of the kynurenine pathway (*kynU*, *kmo*, and *nbaC*) were acquired from eukaryotes through lateral gene transfer [57]. To further investigate this, we conducted a conservation and phylogenetic analysis of the proteins

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Consensus sequences and residue conservation of proteins involved in the anthranilate pathway (TDO, KFA, AFMID, KYNU). The residues that show more than 90 % of conservation are displayed as a consensus sequence. TDO sequences n = 22, KFA sequences n = 15, AFMID sequences n = 17, KYNU sequences n = 25. The gradient colour represents the percentage of conserved residues following the alignment. Grey = less than 50 % conservation. Red gradient = more than 50 % conservation.</figcaption></figure>
associated with the anthranilate pathway.

For TDO (encoded by *kynA*), we identified 41 sequences from 40 distinct organisms (*Delftia acidovorans* SPH-1 having two homologs) within 107 bacterial genomes including 2 *Azospirillum* sp. 4B and *A. baldaniorium* Sp245(Supplementary Fig. S1). Alignment of a representative subset of 22 TDO is presented in Supplementary Fig. S2, with the consensus sequence shown in Fig. 5. The similarity analysis, illustrated by a color gradient indicating the proportion of residue conservation in this dataset, revealed remarkable conservation among various organisms (Fig. 5). Specifically, 41.3 % and 21.8 % of all sites were conserved by at least 70 % and 90 % of taxa, respectively. Besides the well-characterized catalytic sites, the consensus sequence identified a strongly conserved motif (SGFQS) at positions 161–165 and conserved residues at positions 284–311 (Fig. 5- Supplementary Fig. S2). Concurrently, a search for indoleamine 2,3-dioxygenase (IDO), which exhibits similar activity to TDO [65], yielded a positive result only in *Gemmatimonas aurantica*.

For KYNU, we identified 45 sequences in our 107 bacterial genomes subset (Supplementary Fig. S1). A representative subset of 25 was aligned in Supplementary Fig. S3, with the consensus sequence depicted in Fig. 5. The color gradient depicting residue conservation highlighted an intermediate level of preservation throughout the protein sequence. Specifically, 28.1 % and 12.3 % of all sites were conserved by at least 70 % and 90 % of taxa, respectively. In addition to known critical residues, the consensus sequence highlighted an invariant pattern (GNSLG motif) at positions 100–104 and conserved residues between positions 207–212. A consistently invariant 9-amino acid motif (YKXXNXGPG) at position 318–326 was also noted (Supplementary Fig. S3).

For KFA (encoded by *kynB*), 15 homologous sequences were analysed (Supplementary Fig. S1). The BLASTP alignment (Fig. 5 - Supplementary Fig. S4) demonstrated substantial conservation, with 41.1 % and 22.4 % of all sites conserved by at least 70 % and 90 %

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Evolutionary history of KYNU.</figcaption></figure>

The evolutionary history of KYNU was inferred by using the Maximum Likelihood method and Poisson correction model. The tree with the highest likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the BioNJ method to a matrix of pairwise distances estimated using the Poisson model. This analysis involved 45 protein sequences. There was a total of 199 positions in the final dataset. Evolutionary analyses were conducted in MEGA X. The NJ tree (bootstrap = 1000) displays the same topology, except that: (i) sequences of *Deinococcus radiodurans* are part of clade 3a, (ii) the clade 3b is more basal than clade 3a and (iii) the KYNU sequence of *Chlorenflexus auranticus* is more basal than those of the *Actinobacteria* group.
of taxa, respectively. Notably, residues between positions 112–117, 145–158, 189–198, and 204–210 were highly conserved, in addition to key catalytic residues.

In contrast, AFMID (encoded by *afmid*), showed a more variable conservation pattern. Of 17 homologous sequences extracted and aligned (Supplementary Fig. S1), the similarity analysis indicated a patchier conservation across the AFMID polypeptide chain compared to the proteins analysed above (Fig. 5 - Supplementary Fig. S5). Specifically, 19.7 % and only 6.7 % of all sites were conserved by at least 70 % and 90 % of all taxa, respectively. Nonetheless, certain residues were highly conserved, such as YG at positions 75–76, D-84, G-138, Y-146, P-162, D-257, L-278 and G-324.

In conclusion, the similarity analysis revealed variability in the conservation of polypeptides involved in the anthranilate pathway, with TDO and KFA exhibiting higher conservation levels compared to AFMID throughout our subset representative of the bacterial kingdom.

### 3.5. The phylogeny of anthranilate pathway genes reflects a complex evolutionary history

The phylogenetic relationships among the TDO, KFA, AFMID, and KYNU proteins were examined using Rooted Maximum Likelihood trees constructed from sequences obtained via multiple sequence alignment (Gblock program) (see Supplementary Fig. S6-S8).

The phylogenetic analysis of TDO (encoded by *kynA*) reveals five distinct clades. The first clade includes *Bacilli* (Supplementary Fig. S6 - cluster 1 - root). Strangely, the second clade encompasses genetically unrelated taxa, including *Xanthomonas*, (Supplementary Fig. S6 - cluster 2). The third clade groups proteobacterial sequences, including those of *Niveispirillum irakense* and *Nitrospirillum amazonense* (*Alpha-proteobacteria*). Their separation from other *proteobacteria* (Supplementary Fig. S6 - cluster 5a) suggests a distinct evolutionary history for sequences of these strains. *Deinococcus* sequences clusters in clade 4 with those of *Actinomycetes*, except for sequences of *Cellulosimicrobium cellulans* and *Arthrobacter phenanthrenivorans* which are part of the aforementioned clade 2 (Supplementary Fig. S6). Sequences from *Actinomyces* strains exhibit significant divergence compared to other strains, suggesting a more rapid evolutionary rate (Supplementary Fig. S6). The fifth clade encompassed sequences from the *proteobacteria* phylum and is further subdivided into two large subclades: one comprising most of *alpha-proteobacteria*, including all *Azospirillum* sequences (Supplementary Fig. S6 - cluster 5a), and the other dominated by *beta-proteobacteria* and *Pseudomonas* (Supplementary Fig. S6 - cluster 5b).

Overall, the TDO phylogenetic tree largely aligns with established taxonomic classifications, indicating that TDO-encoding genes are typically vertically transmitted with some exceptions.

The phylogenetic tree of KYNU reveals a well-supported pattern of clade distribution (Fig. 6). The first clade contains *Bacilli* sequences (Fig. 6 - cluster 1 - root). The second one comprises sequences from *Flavobacteriia*, *Xanthomonadales*, *Myxococcus xanthus*, and *Verrucosispora maris* (Fig. 6 - cluster 2). Remarkably, within clade 2, KYNU consistently co-localizes with KMO and HAD, while TDO and KFA/AFMID are encoded elsewhere in genomes, a gene association pattern that does not match taxonomic links (Figs. 3 and 6). These results are consistent with previous findings [57], which suggested that the acquisition of the *kynU-kmo-nbaC* gene cluster in *Xanthomonadales*, *Myxococcus xanthus*, and *Flavobacteriia* likely originated from lateral gene transfer from eukaryotes, as these sequences clustered with eukaryotic sequences [57]. Based on our phylogenetic analysis, it is proposed that *Verrucosispora maris* may have acquired this gene cluster in a similar way from a eukaryote or from a bacterial donor having acquired such sequences. Because of the divergent patterns of gene associations for the kynurenine pathway, strains of cluster 2 should be distinguished on the basis of those possessing the kynurenine pathway (via the KMO route) and those possessing the anthranilate pathway. The third clade encompasses *Actinobacteria* and *Proteobacteria*, further divided into four subclusters: the first (Fig. 6 - cluster 3a) encompasses most of *Alpha-proteobacteria*, including *Azospirillum* sp. 4B; the second (cluster 3b) includes strains from various taxonomic origins (e.g. *Sphingomonadales*, and *Gemmatomonadales*) alongside *Nitrospirillum amazonense*; the third (Fig. 6 - cluster 3c) groups mainly strains from the *Rhizobiales* family; and the fourth (Fig. 6 - cluster 3d) encompasses *Gamma-proteobacteria* (notably *Pseudomonadales*) and all *Beta-proteobacteria*. *Deinococcus* sequences are split into two distinct groups: *Deinococcus geothermalis* shares a more recent common ancestor with *Bacilli*, while *Deinococcus radiodurans* is associated with *Alpha-proteobacteria* (Fig. 6 - cluster 3), indicating a dual origin for KYNU in these strains.

Overall, KYNU exhibits a more complex evolutionary history compared to TDO, with phylogenetic patterns not aligning with bacterial taxonomy and suggesting multiple lateral gene transfer events.

AFMID sequences are categorized into three main clades (Supplementary Fig. S7). The first clade includes sequences from *Streptomyces* spp. and *Myxococcus xanthus* strains. The second clade encompasses the AFMID sequence from *Frankia discrariae* which is distinct from the *Actinomycetes* clade and aligns with sequences from *Alcaligenes faecalis* (Supplementary Fig. S7). The third clade covers all *Alpha-proteobacteria* AFMID sequences, including that from *Azospirillum* sp. 4B. Subcluster 3a is characterized by the presence of most of the sequences in the rhizobiales family. This tree highlights a common origin of AFMID for *Alpha-proteobacteria* and *Deinococcus radiodurans* (i.e. similarly to KYNU) (Fig. 6).

In contrast, KFA proteins from *Bacilli* and *Deinococcus geothermalis* (Supplementary Fig. S8 - clade 1) share a probable common origin with their homologs in *Beta-proteobacteria* (Supplementary Fig. S8 - clade 2). Additionally, KFA sequences from *Pseudomonas* seem to derive from the last common ancestor of *Burkholderiaceae*, analogous to TDO and KYNU. These results suggest that KFA might be a more recent acquisition than AFMID. The cyclase-like protein domain likely originated from the exaptation of a cyclase-like enzyme with a different catalytic function. This hypothesis is supported by the fact that hydrolase domain-containing enzymes (the potential oldest kynurenine formamidase isoform) are shared by bacteria and eukaryotes, unlike cyclase domain-containing enzymes, which are present only in bacteria and likely spread via lateral gene transfer.

Overall, the phylogenetic analysis reveals the complex and divergent evolutionary trajectories of anthranilate pathway enzymes
across the bacterial kingdom. Genes encoding TDO and KYNU appear to be vertically inherited punctuated by lateral transfer events, while kynurenine formamidase (KFA/AFMID) phylogeny reflects evolutionary histories not directly linked to taxonomy. The KYN protein phylogeny underscores a conservative clade that includes most *Beta-proteobacteria*, suggesting strong conservation of the acquired gene cluster function and genomic organization within this bacterial phylum. Sequences from *Pseudomonadales* consistently cluster with those of *Burkholderiales*, indicating that *kynA*, *kynB*, and *kynU* genes in these genera may be inherited from a common ancestor. Finally, we propose utilizing KYNU phylogeny as a distinguishing feature to differentiate bacterial strains with the kynurenine pathway (via the KMO route) from those with the anthranilate pathway (Fig. 6).

### 3.6. The anthranilate pathway is specific to all Azospirillum strains of the lipoferum clade

In this study, we noted that the anthranilate pathway was often found in rhizospheric strains, particularly in PGPR. Among PGPR, *Azospirillum* is a rhizobacterial genus from *Alpha-proteobacteria* extensively documented for its ability to establish cooperative relationships with plants, making it a key genus for enhancing the growth and yield of non-leguminous crop species [66].

A comprehensive analysis of the proteins of the Trp catabolism pathways was performed on a set of 26 strains representative of the *Azospirillum* genus. As described previously for the bacterial kingdom, two mutually exclusive Trp catabolic routes span the *Azospirillum* genus. A Maximum Likelihood Phylogenetic Tree was inferred from concatenated RpoD-RecA-GyrA sequences to represent the taxonomy of the *Azospirillum* genus (Fig. 7). On one hand, certain strains had the *ipdC/ppdC* gene (auxin pathway), such as *A. baldaniorum* Sp245 (Sp245) and related strains for which auxin production from Trp has been well characterized [26,61,67–69]. On the other hand, *Azospirillum* sp. 4B (4B) that produces a very small amount of auxin [26] possessed the complete anthranilate pathway. Despite its limited auxin production, 4B can enhance shoot and root dry weights, alter secondary metabolite profiles, stimulate Trp release in rhizodeposits, and activate systemic responses in its host plants, albeit with minimal effects on root architecture compared to Sp245 [70]. The precise underlying phytostimulation mechanism of 4B remains to be determined. Moreover, it is really interesting to notice that 4B and Sp245 possess a single and distinct Trp degradation pathway in their genomes.

The phylogenetic tree generated is broadly consistent with the one derived from *rpoD*, a marker that was suggested to be more relevant than 16S rDNA for *Azospirillum* [71]. Two main clades can be distinguished: a first clade, the "brasilense" clade that includes *A. brasilense*, *A. baldaniorum*, *A. argentinense*, *A. soli* and *A. rugosum* species (shown in purple in Fig. 7), and a second clade, the "lipoferum" clade that gathers *A. lipoferum*, *A. oryzae*, *A. palustre*, *A. melinis* and *A. zeae* species (shown in red in Fig. 7). As previously reported [66], two species, *A. halopraeferens* and *A. fermentarium*, do not cluster within these two clades. This prompted us to use

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Maximum Likelihood taxonomic tree of the *Azospirillum* genus and distribution and organisation of *kynUAB* genes or auxin genes.</figcaption></figure>

The concatenated RpoD, RecA and GyrA sequences were used to deduce a phylogenetic tree by maximum likelihood. Red labels = clade comprising strains of *A. lipoferum* (named clade *lipoferum*). Purple labels = clade including *A. brasilense* strains (named clade *brasilense*). Plant-linked isolate refers to a strain isolated from roots, leaves, rhizosphere or cultivated soil. The *nbaC* gene encoding HAD is present in the genome of *A. lipoferum* Sp59b but is not indicated.
Overall Genome Relatedness Indexes to check their affiliation to the *Azospirillum* genus by calculating ANI (using https://jspecies ribohost.com) and AAI (using http://enve-omics.ce.gatech.edu/aii). The results confirmed their genetic distance from the two major clades, yet the values obtained (>74 % for ANIb and >65 % for AAI) were consistent with their affiliation to the *Azospirillum* genus [72]. By performing similar analyses on the strain CRT1 (originally affiliated to the *lipoferum* species but clustering with the *palustre* species in all trees), it was found that this strain indeed belongs to the *palustre* species (97.71 % for ANIb and 97.83 % for AAI). The analyses also revealed that strain 4B, originally affiliated to the *lipoferum* species did not belong to this species (88.75 % for ANIb between 4B and the type strain *A. lipoferum* Sp59b), nor to any described species.

Regarding to the distribution of genes involved in the anthranilate pathway (identified via BLASTP, Supplementary Fig. S1), it appeared that *kynA*, *kynU* and *afmid* consistently co-occurred in some strains of the *Azospirillum* genus, mostly belonging to the *lipoferum* clade (Fig. 7). Conversely, *kyn* genes were absent from strains of the *brasilense* clade, with the exception of *A. canadense* (Fig. 7). The kynureninase sequences (KYNU) showed 91 %–97 % homology within the *lipoferum* clade while lower homology (58 %) is observed between the *KYNU* sequences of the *lipoferum* clade and *A. halopraeferens*. The distribution of TDO sequences mirrors this pattern, with high homology within the *lipoferum* clade (94–98 %) and reduced conservation in phylogenetically distant strains (e.g. *A. canadense* (88 %), and *A. halopraeferens* (65 %)). A similar trend was observed for kynurenine formamidase (*AFMID*), with homology ranging from 83 % to 95 % within the *lipoferum* clade, 68 % with *A. agricola*, and 87 % with *A. canadense*. In particular, a cyclase-type KFA sequence has been identified in the *A. halopraeferens* strain lacking AFMID, one of the two species phylogenetically distant from the main clades (Fig. 7).

Analysis of the genetic organization of the *kyn* genes showed that *kynU*, *kynA*, and *afmid* exhibit a conserved arrangement at the same locus across all *Azospirillum* except for *A. agricola* where an unrelated gene is inserted between *kynA* and *afmid* (Fig. 7). This gene insertion could potentially impact the functioning of the anthranilate pathway. Moreover, short overlaps between ORFs, as those observed in strain 4B, were not systematically conserved. *Azospirillum* genomes have a complex architecture, being composed of one chromosome (representing about half the genome size) and several plasmids, some being larger than 0,5 Mb and being considered as chromids [26]; interestingly, the *kyn* genes appeared to be always located on a chromid in all *Azospirillum* strains.

When placed back into the pan bacterial kingdom phylogenetic analysis performed on proteins of the anthranilate pathway, all *Azospirillum* sequences clustered within the *Alpha-proteobacteria* group, with the sole exception of those from *A. halopraefrens* sequences (data not shown).

Additionally, no homolog of KMO was identified in strains of the *Azospirillum* genus while HAD was found solely in *A. lipoferum* Sp59b. As a consequence, the KYN proteins found in the *Azospirillum* genus cannot contribute to NAD biosynthesis. Alternatively, the complete aspartate pathway (the alternate pathway to quinolinate and NAD biosynthesis, Fig. 1) is present in all *Azospirillum* genomes (data not shown).

### 3.7. The anthranilate and auxin pathways never co-occur within the *Azospirillum* genus and their presence seems to correlate with developmental impacts on plants

Homologs of indolepyruvate/phenylpyruvate decarboxylase, encoded by *ipdc/ppdc* and critical for IAA synthesis via the IPyA pathway, were identified in the genomes of most strains belonging to the *brasilense* clade, except in *A. canadense* and *A. rugosum* (Fig. 7). Consistent with existing literature, enzymes from the IPyA pathway were absent from strains of the *lipoferum* clade [26]. Moreover, the distribution analysis of all Trp catabolism enzyme genes underlined that both pathways are mutually exclusive within *Azospirillum* strains as in most of the bacterial kingdom (Fig. 7). Given that the acquisition of *ipdc/ppdc* has been described as a recent event in the *Azospirillum* genus [62] potentially autapomorphic within this genus, it is plausible that the lateral gene transfer of *ipdc/ppdc* coincided with the loss of *kyn* genes.

This mutual exclusivity may be attributed to a competition for Trp depletion, antagonistic roles of these pathways, functional redundancy (in the cell functioning or on their host) or the superior function of *ipdc/ppdc* mediated function over the one of the anthranilate pathway. For instance, auxin synthesis by *A. baldaniorum* Sp245 has been identified as the primary mechanism underlying its promotion of root growth, whereas strains such as 4B and *A. palustre* CRT1, which possess the anthranilate pathway, exhibit more limited effects on plant root architecture [70,73,74]. To date, the role of the bacterial anthranilate pathway in plant-bacteria interactions has not been explored. Overall, this analysis underscores the existence of two distinct Trp catabolism pathways within the *Azospirillum* genus.

In parallel, genes encoding aromatic amino acid decarboxylase, associated with the tryptamine pathway, were identified in *A. thermophilum* and *A. halopraeferens*. No additional Trp degradation pathway genes were detected in *Azospirillum*.

Interestingly, certain *Azospirillum* strains, including *A. thiophilum*, *A. rugosum*, *A. fermentarium*, *A. cavernae*, *A. doebereinerae* (Fig. 7), appear to lack all known Trp catabolic pathways. Except for *A. doebereinerae*, these strains were isolated from non-plant-associated environments (e.g., karst caves, hot springs, fermenters, or oil-polluted soils), and their plant growth-promoting potential has not yet been evaluated. Conversely, the majority of strains of this study (90 %) which were isolated from plant-associated environments (interior, surface, or rhizosphere), harbored either the anthranilate or IPyA pathway (Fig. 7). Two exceptions (e.g. *A. griseum* and *A. ramasamyi*) were isolated from lake water and bovine dung, respectively, suggesting that their natural habitats may not have involved direct contact with plants.

This study is based on *in silico* analyses of genomic data related to the catabolism of tryptophan. While our findings suggest that *Azospirillum* strains tend to harbour genes enabling either auxin (*brasilense* clade) or anthranilate/kynurenic acid production (*lipoferum* clade), no functional validation was performed. Therefore, the presence of these genes does not necessarily reflect their expression or metabolic activity under physiological conditions, and especially during their interactions with plants. Further experimental studies,
including functional assays and metabolite profiling, are required to validate these predicted capabilities and to better understand their biological relevance in the *Azospirillum*-plant interactions.

## 4. Conclusion

In summary, Trp catabolism appears to be largely conserved among *Azospirillum* strains involved in associative symbiosis with host plants. Plants release Trp, serving as a substrate for the two bacterial pathways identified in *Azospirillum*. However, two distinct strategies seem to have evolved within this genus: one leading to auxin biosynthesis, which is associated with enhanced root branching, and the other producing anthranilate, with effects on host plant physiology that require further investigation.

## Funders and Grants ID

This work was supported by a doctoral contract funded by the "Ministère de l'Enseignement Supérieur et de la Recherche". The experimental work was carried out with the support of the French National Centre for Scientific Research (CNRS) and by University Lyon.

## CRediT authorship contribution statement

**Adrian Lautrey:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Laurent Legendre:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. **Florence Wisniewski-Dyé:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Claire Prigent-Combaret:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

A.L. received a fellowship from "Ministère de l'Enseignement Supérieur et de la Recherche". The LABGeM (CEA/Genoscope & CNRS UMR8030), the France Génomique and French Bioinformatics Institute national infrastructures (funded as part of Investissement d'Avenir program managed by Agence Nationale pour la Recherche, contracts ANR-10-INBS-09, ANR-11-INBS-0013 and ANR-21-ESRE-0048) are acknowledged for support within the MicroScope annotation platform.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2025.e44009.

## Data availability

Data are available in Supplementary Material. For requesting other information, please write to the corresponding author (C. Prigent-Combaret).

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