INTRODUCTION
The sensing of environmental signals allows bacteria to adapt to changes that occur in their ecological niches or during host colonization (1). The number of signal transduction systems encoded in the genomes of microorganisms with a complex lifestyle is particularly high (2, 3), suggesting that these microbes respond to a particularly broad range of physical, chemical, and biological stimuli. In this context, plants and their associated microorganims are holobionts (4–6), and the interaction between plants and their microbiota, as well as between different phyto-microorganisms, is highly dynamic and controlled by the concerted action of multiple signal molecules (1, 4, 7). This intricate communication shapes the structure of the plant microbiota (4, 6, 8).
The chemical diversity of plant signals that modulate bacterial physiology and metabolism is high, including amino and organic acids, sugars, peptides, aromatic acids, polyamines, phenolic compounds, fatty acids, phytohormones, inorganic ions, and volatiles (1, 4, 7, 9–12). Given the complexity of this chemical language, plant-associated bacteria (PAB) possess multiple mechanisms that permit the generation of responses to the signals sensed (1, 3, 7, 13). For example, the important role of plant-bacteria communication is reflected by the fact that PAB have, on average, twice as many chemoreceptors compared to bacteria that do not interact with plants (2). Nonetheless, the molecular mechanisms by which this plant-bacteria communication occurs remain largely unknown (6, 7).
A number of key plant signaling molecules were shown to be of great relevance to establish plant-bacteria interactions, including cytokinins (7, 14), ethylene (15), gibberelins (16, 17), salicylic acid (1, 15), abscisic acid (15, 18), auxins (19, 20), ɣ-aminobutyric acid (7, 21), and strigolactones (15, 22). The auxin indole-3-acetic acid (IAA) is a key phytohormone that coordinates plant growth and development, as well as plant responses to a broad range of biotic and abiotic stresses (23–25). In addition, IAA is an essential signal molecule in the interaction between plants and microbes. Thus, next to its production by plants, most PAB were found to produce significant amounts of IAA (26, 27), indicating that IAA-mediated plant-bacteria communication is bidirectional and as such highly complex. IAA production by beneficial PAB typically results in plant growth promotion (15, 19), but alterations in IAA homeostasis in the plant by phytopathogenic bacteria are closely associated with virulence-related processes such as tumor formation, epiphytic colonization, or modulation of plant defenses (20).
In addition to its role as an inter-kingdom signal, IAA is an important bacterial signal molecule that regulates biofilm formation, primary and secondary metabolism, auxin catabolism, virulence factor production, chemotactic responses, and plant host colonization, among other processes (19, 20, 28–33). However, the molecular mechanisms underlying these regulatory processes have been identified in only a few cases, such as the IAA-mediated virulence suppression through the inhibition of phytotoxin biosynthesis (28). Likewise, the regulation of the IAA catabolism depended on several IAA-responsive transcriptional regulators (30). Additionally, we have recently identified a chemoreceptor that mediates IAA chemoattraction in a beneficial PAB (31). Notably, bacterial signal transduction receptors can also be stimulated by the binding of signal-loaded solute-binding proteins (13) and several IAA-responsive solute-binding proteins have been identified (32, 34) that may potentially exert a role in signal transduction.
We have recently established the rhizosphere biocontrol agent
The inactivation of IAA synthesis resulted in important transcriptional changes in
RESULTS
IAA treatment causes important changes in transcript levels
To investigate the effects of IAA on the global transcriptome of
Fig 1
The expression profile of the andrimid biosynthetic gene cluster of
RNA-seq data revealed that IAA treatment caused important transcriptional changes in A153. Statistically significant differentially expressed genes (DEGs) were selected based on a fold-change magnitude of log2 greater than 1.5 and an adjusted
Fig 2
The transcriptome of
TABLE 1
Selected differentially expressed genes regulated in response to 1 mM indole-3-acetic acid
Locus no. | Preferred gene name | Known or predicted function | Log2 fold change | COG class |
---|---|---|---|---|
Upregulated | ||||
Energy production and conversion | ||||
| FMNH2-dependent alkanesulfonate monooxygenase | 6.0 | C | |
| NADPH quinone reductase | 3.1 | S | |
| NADPH-dependent FMN reductase | 4.3 | S | |
Amino acid transport and metabolism | ||||
| Acetolactate synthase | 4.1 | EH | |
| Pyrroline-5-carboxylate reductase. L-Pro metabolism | 2.0 | E | |
| Glutamate/phenylalanine/leucine/valine/L-tryptophandehydrogenase | 1.6 | E | |
| LysE family transporter | 1.9 | E | |
| Threonine/serine exporter | 3.8 | E | |
| Anthranilate synthase component | 2.5 | E | |
| D-amino acid dehydrogenase | 3.1 | E | |
Carbohydrate metabolism and transport | ||||
| Putative sugar transporter | 5.4 | G | |
| Phosphoenolpyruvate phosphomutase | 4.6 | G | |
| Carbohydrate periplasmic-binding protein | 5.5 | G | |
| Glycosyl transferase | 3.2 | G | |
Transcription | ||||
| Transcriptional regulator | 1.9 | K | |
| LysR type transcriptional regulator | 3.4 | K | |
| LysR type transcriptional regulator | 1.9 | K | |
– | TetR type transcriptional regulator | 4.4 | K | |
| TetR type transcriptional regulator | 2.2 | K | |
| LuxR type transcriptional regulator | 1.8 | KT | |
| LysR type transcriptional regulator | 3.1 | K | |
Cell wall/membrane/envelop biogenesis | ||||
| Porin | 2.1 | M | |
| UTP-glucose-1-phosphate uridylyltransferase | 1.9 | M | |
| Polysaccharide export protein | 1.9 | M | |
| Pyruvyl transferase | 2.2 | S | |
| Glycosyl transferase | 2.5 | M | |
| Glycosyl transferase | 2.3 | S | |
| Glycosyl transferase | 3.1 | G | |
| Hypothetical protein | 2.0 | – | |
| Glycosyl transferase | 2.7 | M | |
| Glycosyl transferase | 2.8 | M | |
| Bacterial sugar transferase | 2.5 | M | |
– | Right-handed parallel beta-helix repeat-containing protein | 2.8 | – | |
| Hypothetical protein | 2.3 | Q | |
| Secretion system permease | 1.7 | V | |
| Mannose-6-phosphate isomerase | 1.5 | GM | |
| Phosphoglucomutase/phosphomannomutase | 1.9 | G | |
| UDP-glucose 4-epimerase | 1.6 | M | |
| Glycosyl transferase | 1.8 | M | |
Cell motility | ||||
| Flagellar protein | 4.0 | N | |
Inorganic ion transport and metabolism | ||||
| Metal efflux RND transporter | 3.7 | P | |
| Potassium transport system | 2.5 | P | |
| Potassium transport system | 5.2 | P | |
– | TonB-dependent receptor | 4.7 | P | |
Secondary metabolites biosynthesis, transport, and catabolism | ||||
| Dabb family protein | 1.9 | – | |
| Polyketide synthase | 1.5 | – | |
| Ester cyclase | 1.9 | – | |
| Non-ribosomal peptide synthetase | 1.6 | – | |
| Dehydratase | 1.5 | – | |
| Dehydratase | 2.1 | – | |
| Hypothetical protein | 1.7 | – | |
Signal transduction mechanisms | ||||
| Diguanylate phosphodiesterase—EAL domain-containing protein | 2.0 | T | |
| Diguanylate cyclase | 1.8 | T | |
– | Diguanylate phosphodiesterase—EAL domain-containing protein | 2.3 | T | |
Intacellular trafficking, secretion, and vesicular transport | ||||
| Multidrug efflux RND transporter permease subunit | 1.9 | U | |
| 3.9 | U | ||
| 4.5 | U | ||
| Hypothetical protein | 4.8 | S | |
| 2.1 | G | ||
| Carbon starvation protein CstA | 1.6 | T | |
Defense mechanisms | ||||
| Macrolide ABC transporter permease | 3.7 | V | |
| Effector protein | 2.5 | S | |
– | Coalicin immunity protein | 2.5 | S | |
– | Multidrug resistance protein | 2.1 | E | |
| Outer membrane permeability protein | 2.5 | S | |
| Universal stress protein | 2.4 | T | |
| Beta lactamase | 2.4 | V | |
Unknown function | ||||
– | Hypothetical protein | 1.6 | – | |
| Hypothetical protein | 2.9 | – | |
| Hypothetical protein | 4.1 | – | |
| Hypothetical protein | 3.3 | – | |
Downregulated | ||||
Biosynthesis of cofactors, prosthetic groups and carriers | ||||
| Oxidoreductase | −2.5 | C | |
| Nitrate reductase subunit | −2.5 | C | |
| Oxidoreductase | −2.7 | C | |
| Glycerol-3-phosphate dehydrogenase | −1.6 | C | |
Amino acid transport and metabolism | ||||
– | Aminotransferase | −2.2 | E | |
| Amino acid ABC transporter permease | −2.7 | E | |
– | Amino acid permease | −2.3 | E | |
| Aspartate/lysine aminotransferase | −2.2 | E | |
Carbohydrate metabolism and transport | ||||
| Sucrose-6-phosphate hydrolase | −2.4 | G | |
| Phospho-beta-glucosidase | −2.5 | G | |
| Transporter permease | −4.8 | EG | |
| Chitinase protein | −1.9 | G | |
Transcription | ||||
– | LysR type transcriptional regulator | −2.9 | K | |
| LysR type transcriptional regulator | −1.7 | K | |
– | TetR type transcriptional regulator | −1.7 | K | |
– | AraC type transcriptional regulator | −2.3 | K | |
| Metal-sensing transcriptional regulator | −1.7 | S | |
| LysR type transcriptional regulator | −2.1 | K | |
Cell motility and secretion | ||||
– | Fimbrial protein | −1.9 | NU | |
| Chemotaxis protein | −2.2 | NT | |
| Chemotaxis protein | −2.3 | NT | |
Inorganic ion transport and metabolism | ||||
– | Periplasmic-binding protein | −2.1 | P | |
| Ion-transport protein | −2.2 | P | |
| Sulfate/thiosulfate ABC transporter permease | −3.3 | P | |
| Sulfite reductase flavoprotein subunit | −2.5 | P | |
| TonB-dependent receptor | −1.7 | P | |
Signal transduction mechanisms | ||||
| Histidine kinase | −1.9 | T | |
| Histidine kinase | −1.7 | T | |
| Histidine kinase | −2.5 | T | |
| Diguanylate cyclase | −2.1 | T | |
Intracellular trafficking, secretion, and vesicular transport | ||||
– | Major facilitator superfamily transporter | −2.0 | EGP | |
– | Major facilitator superfamily transporter | −2.0 | EGP | |
| Colicin uptake protein | −2.0 | U | |
– | Major facilitator superfamily transporter | −3.5 | EGP | |
Defense mechanisms | ||||
| Acid resistance protein | −2.1 | S | |
Unknown function | ||||
– | Hypothetical protein | −2.4 | – | |
– | Hypothetical protein | −2.1 | – | |
– | Putative glutathione-dependent formaldehyde-activating enzyme | −3.8 | S | |
– | Hypothetical protein | −4.3 | – | |
– | Hypothetical protein | −4.8 | – |
The complete list of differentially expressed genes is provided in Table S1.
C, energy production and conversion; E, amino acid transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; K, transcription; M, cell wall/membrane/envelope biogenesis; N, cell motility; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms.
Differentially expressed gene also identified in response to 0.25 mM indole-3-acetic acid treatment.
Large functional diversity of IAA-regulated genes
Using clusters of orthologous genes (COGs) (41), DEGs were classified into 20 functional categories (Fig. 2D; Fig. S2; Tables S1 and S2), indicative of the complexity of the IAA-mediated responses. In response to 0.25 mM IAA, most populated categories were amino acid transport and metabolism (15%), carbohydrate metabolism and transport (8.3%), energy production and conversion (11.6%), transcription (6.7%), and inorganic transport and metabolism (8.3%) (Fig. S2; Table S2). Alternatively, amino acid transport and metabolism (8.8%), carbohydrate metabolism and transport (7.6%), energy production and conversion (6.5%), transcription (13.1%), coenzyme transport and metabolism (4.9%), cell wall/membrane biogenesis (6.3%), inorganic transport and metabolism (4.8%), and intracellular trafficking and secretion (6.2%) were the most populated functional categories in response to 1 mM IAA, with 20.2% of the DEGs being of unknown function (Fig. 2D; Table S1).
To validate our RNA-seq data, we performed quantitative real time PCR (RT-qPCR) assays of a selection of 14 DEGs belonging to 9 functional categories (e.g., metabolism and transport of organic and inorganic metabolites, cell wall and membrane biogenesis, transcription, signal transduction, and intracellular trafficking). The results correlated well with the RNA-seq data (Fig. S3; Tables S1 and S2).
IAA treatment caused large alterations in the metabolic capabilities of
Given the large number of DEGs with implications for cellular metabolism and transport of organic and inorganic nutrients (Fig. 2D; Fig. S2; Tables S1 and S2), we investigated the effect of IAA on the metabolic capabilities of A153. First, to identify compounds that may serve as carbon and nitrogen sources, we analyzed growth using commercial Biolog Phenotype MicroArray plates PM1 and PM3B, each containing 95 potential carbon and nitrogen sources, respectively. These metabolites included sugars, sugar phosphates, amino and organic acids, alcohols, purines, pyrimidines, dipeptides, among others. Growth assays showed that A153 is able to use 65% and 77% of the tested metabolites as sole carbon and nitrogen source, respectively (Table S5). Subsequent analyses showed that exposure to 1 mM IAA altered the metabolic capacities for the utilization of 37% of the carbon sources tested (Fig. 3A; Fig. S4; Table S5). Particularly, the metabolism of amino acids, dipeptides, as well as that of aliphatic and aromatic organic acids was mainly affected. In contrast, no significant alteration was observed in the metabolism of other metabolites such as sugars, sugar derivatives, purines, or pyrimidines (Table S5). Alternatively, IAA treatment altered the metabolism of 22% of the nitrogen sources tested, primarily amino acid metabolism. IAA also slightly modulated growth on the pyrimidines thymidine, uracil, and uridine as sole N-sources (Fig. 3B; Fig. S5; Table S5), which contrasted with the above growth experiments when used as sole C-sources. In most cases, IAA decreased the growth rate, with the exception of L-Ala as sole carbon source, and L-Ile, L-Leu, L-Thr, and L-Val as sole nitrogen sources (Fig. 3; Fig. S4 and S5; Table S5).
Fig 3
Indole-3-acetic acid affects the metabolism of different nutrients as a sole carbon (A) and nitrogen (B) sources in
Since most of the compounds with an altered metabolism in the presence of IAA are present in root exudates (42, 43), we investigated the effect of IAA on the growth kinetics of A153 in maize root exudates, which are rich in amino and organic acids (44). The results revealed that IAA treatment caused a reduction in A153 growth in root exudates (Fig. S6).
IAA modulates the expression of the AaeXAB efflux pump to confer resistance to toxic aromatic acids and IAA in
Among the genes with the highest levels of induction in response to IAA were those encoding the efflux pump AaeXAB, with induction values ranging from 4.8- to 11.2-fold and 15.3- to 28.2-fold in response to 0.25 mM and 1 mM IAA, respectively (Table 1; Tables S1 and S2). These data were subsequently confirmed by RT-qPCR (Fig. 4A). The AaeXAB efflux pump was identified in
Fig 4
Indole-3-acetic acid (IAA) regulates the expression of the AaeXAB efflux pump to control resistance to high levels of IAA in
To investigate whether the AaeXAB pump could be acting as a protective mechanism against high IAA concentrations, we evaluated the growth of the
The expression of the
IAA enhances ampicillin resistance in
Several genes that were shown to be involved in antibiotic resistance in other bacterial species were induced in A153 in response to IAA (Table 1; Tables S1 and S2), including those encoding the OmpC porin (50), the multidrug transporter permeases SanA (51) and MdtB (52), the multidrug transporter MacB (53), a multidrug resistance protein (AWY96_RS15045) (54), capsular polysaccharide (CPS) biosynthesis proteins (55), and a β-lactamase (AWY96_RS20405) (56). To investigate the effect of IAA on antibiotic resistance in A153, we determined the MIC values of various antibiotics that operate with different mechanisms of action, namely, ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, streptomycin, rifampicin, and tetracycline. We found that IAA treatment enhances resistance to gentamicin and kanamycin (Table S6), two aminoglycoside antibiotics that inhibit protein synthesis by binding to the 30S ribosomal subunit (57). Subsequent experiments revealed that IAA significantly increased resistance to ampicillin in minimal medium agar plates by at least an order of magnitude (Fig. 5), which correlates with the increased expression of the β-lactamase encoding gene
Fig 5
Indole-3-acetic acid (IAA) increases ampicillin resistance in
Because antibiotic tolerance, defined as the ability of bacteria to survive exposure to antibiotics, may not result in changes in MIC values (58), we evaluated the effect of IAA on the survival of A153 cells in exponential phase of growth after treatment with the high concentrations of ampicillin, gentamicin, kanamycin, nalidixic acid, streptomycin, and rifampicin. No differences were observed in the presence and absence of IAA, collectively indicating that this auxin does not induce tolerance to the antibiotics tested.
IAA treatment reduces c-di-GMP levels altering motility and biofilm formation
The second messenger c-di-GMP plays an important role in the transition from a motile to a sessile lifestyle in bacteria (59, 60). The identification of several DEGs involved in c-di-GMP turnover and motility (Table 1; Tables S1 and S2) encouraged us to analyze the role of IAA in the modulation of several c-di-GMP-regulated phenotypes. First, we conducted swimming motility assays and observed a significant increase in motility in response to increasing IAA concentrations, causing a ~30% increase in the swimming diameter in the presence of 1 mM IAA (Fig. 6A). Second, we found that IAA inhibited biofilm formation, resulting in a total inhibition at a concentration of 1 mM IAA (Fig. 6B). These motility and biofilm phenotypes are in agreement with a IAA-mediated decrease in c-di-GMP levels. To further investigate this issue, we quantified the global c-di-GMP levels in A153 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that IAA reduced c-di-GMP levels in a concentration-dependent manner, causing a ~31% and ~64% decrease in the presence of 0.25 mM and 1 mM IAA, respectively (Fig. 6C).
Fig 6
IAA treatment lowers c-di-GMP levels to promote motility and inhibit biofilm formation in
IAA exposure caused an upregulation of a ~45 kbp biosynthetic gene cluster (
IAA exposure increases sensitivity to a capsule-dependent phage
We previously isolated the bacteriophage ɸMAM1 that infects
Fig 7
IAA treatment promotes phage attachment to
TrpRA153 is an auxin-binding transcriptional regulator whose expression is upregulated by IAA
Numerous transcriptional regulators encoding genes were up- and downregulated in response to IAA (Fig. 2D; Fig. S2; Tables S1 and S2). Among these genes, we found that
Fig 8
Isothermal titration calorimetry studies of the binding of different ligands to TrpRA153 of
To investigate whether IAA and IPA compete with L-Trp for binding to TrpRA153, we conducted competitive binding assays. We first analyzed the capacity of IAA and IPA to compete for binding to TrpRA153 through microcalorimetric titrations. The results revealed that saturating TrpRA153 with either IAA or IPA prevented binding of IPA or IAA, respectively, to the regulator (Fig. S10), indicating that both auxins compete for binding to TrpRA153. Subsequent experiments revealed an absence of L-Trp binding to TrpRA153 in the presence of saturating concentrations of IAA or IPA (Fig. 8). Alternatively, no IAA binding to TrpRA153 was noted in the presence of saturating concentration of L-Trp (Fig. S10). Taken together, these results indicate that IPA and IAA compete with L-Trp for their binding to TrpRA153. This also implies that changes in L-Trp levels interfere with auxin-mediated signaling. To our knowledge, A153 is the only bacterium in which two auxin-binding transcriptional regulators have been identified and future studies will focus on analyzing the ligands and regulatory cascades of the distinct regulators whose expression is modulated by IAA.
DISCUSSION
IAA is a multi-faceted signal molecule that exerts a variety of regulatory functions in phylogenetically distant species. Next to its pivotal role in plant growth and development (23–25), it regulates fungal physiology (67), microalgal growth (68), inflammatory and carcinogenic processes in animals and humans (69, 70), and, as shown here, bacterial metabolism and physiology. Among the signals that play a major role in plant environments, IAA is emerging as a key compound, allowing plant-associated microbes to adapt efficiently to their hosts and to establish interactions with other (micro)organisms in plant niches (18–20, 29, 33). However, we are currently only at the beginning of understanding the mechanisms by which IAA regulates bacterial physiology, metabolism and social behavior.
Previous research revealed an effect of exogenous (71–75) and endogenous (29, 39, 73, 76) IAA on the global transcriptomes of different plant-associated bacteria. These studies demonstrated a role of IAA in the modulation of bacterial transcriptomes, including the differential regulation of genes involved in stress responses (29, 74), nitrogen fixation (76), metabolism (39, 72, 73), as well as pathogenesis and virulence factor production (71, 75, 77). These differential and multifaceted regulatory effects, together with the fact that current research on bacterial auxin signaling has focused mainly on plant pathogenic and nitrogen-fixing bacteria, reinforces the need to investigate IAA signaling pathways in different model microorganisms, as well as to advance in the mechanisms by which IAA exerts its activities.
Our results show that IAA causes remarkable changes in the transcriptome of
Secondary metabolism represents a metabolic burden for bacteria since it diverts energy, precursors, and cofactors from primary metabolism (80, 81). Andrimid biosynthesis requires the amino acid phenylalanine, glycine, and valine as precursors (82), and we found that IAA alters phenylalanine and valine metabolism—an aspect that may modulate andrimid biosynthesis in response to IAA. In addition, the expression of the andrimid operon was found to be regulated post-transcriptionally (36), and we identified four sRNAs encoded in the andrimid operon that were upregulated by IAA (Table S4). We hypothesize that these sRNAs could act as translational repressors of the andrimid operon, and future research will further assess this hypothesis. Several studies have attributed a role for IAA in the regulation of bacterial secondary metabolism, both activating and repressing antibiotic production (28, 37, 83, 84)—suggesting a role for IAA-mediated signaling in allowing bacteria to thrive in complex and highly competitive niches. Notably, additional phytohormones like jasmonic acid and salicylic acid were also shown to modulate antibiotic production in plant-associated bacteria (83, 85).
Plants have evolved several strategies to control IAA homeostasis (86). Analogously to plants, bacteria have developed various mechanisms to control IAA homeostasis and to counteract possible toxic effects of high IAA concentrations, including auxin catabolism (30, 87), the generation of IAA inactive conjugates (19, 88), and auxin efflux (89). Here, we discovered that the efflux pump AaeXAB, in addition to conferring resistance to 4HBA, also confers resistance to high levels of IAA. To the best of our knowledge, MatE is the only so far known bacterial transporter involved in IAA efflux (89), and future studies will analyze the role of the AaeXAB pump in IAA extrusion. 4HBA is a signal molecule that regulates different features of plant-bacteria interaction, including plant defense against pathogens (90), bacterial phytopathogenicity (47, 91, 92), chemotaxis (91, 93), as well as exopolysaccharide (94) and pigment (92) biosynthesis. We showed that IAA has an important effect on the expression of a 4HBA efflux pump, indicative of a cross-talk between IAA- and 4HBA-controlled regulatory circuits. In addition, we also showed that different auxins (e.g., IPA and IAA) and L-Trp compete for binding to TrpRA153, suggesting the existence of a cross-regulation between auxins and amino acids. Together, these findings highlight the complexity of IAA-mediated signaling and illustrate the intricacy of chemical plant-bacteria signaling.
The upregulation of the efflux pump AaeXAB in response to IAA as well as that of genes involved in capsule synthesis and with implications for antibiotic resistance may be indicative of stress. Previous studies have shown that IAA modulates different physiological and metabolic bacterial processes of importance during interaction with plants (19, 20, 33). For example, during plant colonization, bacteria face multiple stresses such as oxidative stress, presence of antimicrobial compounds, and adaptation to specific nutrients (95–98), and IAA has been shown to play a role in adapting to these and other environmental stresses (19, 33). Notably, it has been shown that IAA present in root exudates affects the composition of microbial communities (18) and that plant-associated bacteria exhibit chemotaxis towards IAA (31) and are frequently able to catabolize IAA (30, 99)—highlighting again the biological significance of IAA in mediating plant-bacteria interactions. IAA can be found in the rhizosphere at micromolar concentrations (100, 101), but further studies are needed to determine the IAA concentrations at the microscale in plant niches. By analogy with our data, jasmonic acid, another key phytohormone, was also shown to modulate the physiology of plant-associated bacteria (85).
We showed here that IAA exposure modulates antibiotic susceptibility, which is reminiscent to the observation that IAA increases ampicillin resistance in the biocontrol agent
Since its discovery in the late 1980s, c-di-GMP has emerged as one of the key bacterial second messengers, being involved in the coordination of critical processes such as biofilm formation, motility, cell development, and virulence (59, 60). Although several studies showed that IAA treatment affects biofilm formation (74, 105) and motility (75, 76), the underlying mechanisms remain largely unknown. To the best of our knowledge, we establish here, for the first time, an effect of IAA on c-di-GMP-mediated signaling. IAA decreased c-di-GMP levels which correlated with the increased motility and decreased biofilm-forming capacity observed in this condition. Furthermore, IAA upregulated the expression of the
Conclusions and future perspectives
Our knowledge of the signaling roles of IAA opens possibilities for different biotechnological and clinical applications (69, 109–111), including (i) the biosynthesis of products of clinical and industrial interest, (ii) the construction of biosensors, (iii) anti-virulence therapies, (iv) GSE248473 identificationof antibiotic adjuvants, and (v) microbiome engineering as a strategy to promote plant and animal health. This study advances our knowledge of the mechanisms by which IAA modulates motility, biofilm formation, bacteriophage sensitivity, and resistance to antimicrobials. Given the role of efflux pumps as virulence factors (112), as well as the re-emerging potential of phage therapy to combat multidrug-resistant infections (113), research derived from this work will pave the way for studies aimed at utilizing auxins as anti-virulence agents to combat multidrug-resistant pathogens.
MATERIALS AND METHODS
Strains, bacteriophages, plasmids, oligonucleotides, and culture conditions
Bacteria, phages, and plasmids used in this study are described in Table S7, whereas oligonucleotides are listed in Table S8.
Plasmid DNA was isolated using the NZY-Miniprep kit (NZY-Tech). For DNA digestion, alkaline phosphatase, and ligation reactions, manufacturers’ instructions were followed (New England Biolabs and Roche). DNA fragments were recovered from agarose gels using the Qiagen gel extraction kit. PCRs were purified using the Qiagen PCR Clean-up kit. PCR fragments were verified by DNA sequencing that was carried out at the Institute of Parasitology and Biomedicine Lopez-Neyra (CSIC; Granada, Spain). Transformations and electroporations were performed using standard protocols (114). Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific) was used for the amplification of PCR fragments.
Marker exchange mutagenesis
A deletion mutant defective in
Phenotypic assays
Antagonistic activities against
Collection of root exudates
Maize seeds were sterilized and germinated as described previously (96) and root exudates were collected from 16 germinated seeds as previously indicated (44). Maize root exudates were freeze-dried and stored at −80°C until use.
Growth experiments and antibiotic susceptibility assays
A153 was grown overnight in minimal medium containing 15 mM glucose. Overnight cultures were washed twice and then diluted to an OD600 of 0.02 in minimal medium containing either: (i) 15 mM glucose supplemented with different concentrations of antibiotics, (ii) 15 mM glucose supplemented with different concentrations of 4HBA and/or IAA, or (iii) each of the compounds present in the Biolog (Hayward, CA, USA) compound arrays PM1 and PM3B as sole carbon source and nitrogen sources, respectively. Growth in root exudates was done in 50 mM phosphate buffer in the presence of 10× and 100× maize root exudates, concentrations that correspond to 2.5 and 25 g/L freeze-dried exudates, respectively. Differences in the growth of A153 in the absence and presence of 1 mM IAA were considered when measuring alterations in growth rate, lag phase, and/or when the maximum OD600 reached in growth experiments varied by at least 10%. In all cases, 200 µL of the cultures were transferred into microwell plates, and growth at 30°C was followed on a Bioscreen microbiological growth analyzer (Oy Growth Curves Ab Ltd., Helsinki, Finland).
Minimal inhibitory concentration (MIC) assays were performed in minimal medium supplemented with 15 mM glucose in the presence and absence of 1 mM IAA using a twofold serial dilution test (116). The MIC was established as the lowest concentration of a compound that prevented growth in liquid cultures after 48 h at 30°C. Growth was followed on a Bioscreen microbiological growth analyzer (Oy Growth Curves Ab Ltd., Helsinki, Finland). Alternatively, the effect of IAA on antibiotic resistance was also assessed in solid media. Briefly, serial dilutions of overnight cultures grown in minimal medium in the presence and absence of 1 mM IAA were spot-plated onto minimal medium agar supplemented with antibiotics or both 1 mM IAA and antibiotics (50–150 μg/mL ampicillin, 1–12 μg/mL gentamicin, 3–25 μg/mL kanamycin, 1–5 μg/mL nalidixic acid, 3–25 μg/mL streptomycin, and 5–20 μg/mL rifampicin). Serial dilution plates were allowed to grow overnight at 30°C.
Antibiotic tolerance was assessed in minimal medium supplemented with 15 mM glucose. Briefly, overnight cultures in minimal medium were used to inoculate fresh medium with and without 1 mM IAA to reach an OD600 of 0.1. Cells were then cultured at 30°C until an OD600 of 0.4, at which time the cultures were challenged with different antibiotics (100–400 μg/mL ampicillin, 10 µg/mL gentamicin, 50 µg/mL kanamycin, 10–20 μg/mL nalidixic acid, 50 µg/mL streptomycin, and 10–40 μg/mL rifampicin) for a period from 1 to 3 h. Survival was determined by serial dilution plating comparing the colony counts before and after antibiotic treatment.
Phage adsorption assays
Phage adsorption assays were conducted as described previously (61), with minor modifications. Briefly, an overnight bacterial culture of A153 was adjusted to an OD600 of 0.1 in minimal medium in the presence and absence of 1 mM IAA. After overnight growth, 5 mL cultures were then infected with ɸMAM1 at a multiplicity of infection of 0.01, mixed briefly, and placed on a tube roller at 25°C. A bacterium-free negative control was created by adding the same amount of phage to 5 mL of minimal medium. One-hundred-microliter samples were removed at different times and added to 900 µL of phage buffer [10 mM Tris-HCl, 10 mM MgSO4, 0.01% (wt/vol) gelatin, pH 7.4] containing 30 µL of chloroform. The components were mixed for 5 s and centrifuged at 13,000 ×
c-di-GMP quantification by liquid chromatography-tandem mass spectrometry
A153 cultures grown overnight in minimal medium were used to inoculate 100-mL flasks containing 20 mL of fresh medium with and without 0.25 and 1 mM IAA to an OD600 of 0.1. After 16 h of growth at 30°C, 10 mL samples were harvested by centrifugation at 2,500 ×
RNA extraction, cDNA synthesis, and quantitative real-time PCR analyses
Total RNA was extracted using TRI Reagent (Invitrogen) followed by Turbo DNase treatment (Ambion) and RNA clean-up with RNeasy Mini Kit (Qiagen) according to manufacturers’ instructions. RNA degradation and contamination were assessed by electrophoresis on 2% (wt/vol) agarose gels. The synthesis of cDNA was performed using random hexamers (GE Healthcare) and SuperScript II reverse transcriptase (Invitrogen) in a 25 µL reaction volume with 1 µg of total RNA and incubation at 42°C for 2 h. RT-qPCRs were performed as described previously (36) using primers described in Table S8. RT-qPCR amplifications were performed using the iQ SYBR Green supermix (Bio-Rad) in an MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad) associated with iQ5 optical system software (version 2.1.97.1001). To confirm the absence of contaminating genomic DNA, control PCRs were carried out using no RT cDNA samples as templates. Melting curve analyses were conducted to ensure the amplification of a single product. The relative gene expression was calculated using the critical threshold (ΔΔCt) method (118) using the
RNA-seq and data analysis
RNA sequencing was done at the GENYO Research Center (Granada, Spain). Prior to preparation of the RNA library, ribosomal RNAs were removed from the samples using the RiboZero Magnetic Kit (Epicentre; Ref. MRZGN126) following the manufacturers’ instructions. Subsequently, samples were processed with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) following the provided sample preparation guide. The final library (adapter and index included) was validated using the DNA-specific chip Agilent DNA 1000. The final products were fragments between 265 and 300 bp. RNA-seq libraries were sequenced on the Illumina NextSeq 500 sequencer. Initial quality control checks were carried out using FastQC software on the raw sequences (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Read mapping and quantification were carried out with the EDGE-pro program (119). Sequences were aligned with the reference genome
Protein overexpression and purification
Isothermal titration calorimetry
Measurements were made using a VP-ITC microcalorimeter (MicroCal, Inc., Northampton, MA) at 25°C. TrpRA153 was dialyzed into 5 mM Tris, 5 mM pipes, 5 mM MES, 150 mM NaCl, 10% (vol/vol) glycerol, pH 8.0. Alternatively, AaeR-LBD was dialyzed into 20 mM HEPES, 150 mM NaCl, 2 mM DTT, 5% (vol/vol) glycerol, pH 7.5. Proteins at 50–100 µM were placed into the sample cell and titrated with 6.4–12.8 µL aliquots of 1–5 mM ligand solutions made up in the corresponding dialysis buffers. In all cases, the mean enthalpies measured from the injection of the ligand in the buffer were subtracted from raw titration data before data analysis with the ORIGIN software (MicroCal).
Differential scanning fluorimetry
Assays were performed on a MyiQ2 real-time PCR instrument (Bio-Rad), as previously described (37). Ligands from the PM1, PM2A, PM3B, PM4A, and PM5 compound arrays (Biolog, Hayward, CA, USA) were dissolved in 50 µL of Milli-Q water, which, according to the manufacturer, corresponds to a concentration of 10–20 mM.
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Abstract
ABSTRACT
The communication between plants and their microbiota is highly dynamic and involves a complex network of signal molecules. Among them, the auxin indole-3-acetic acid (IAA) is a critical phytohormone that not only regulates plant growth and development, but is emerging as an important inter- and intra-kingdom signal that modulates many bacterial processes that are important during interaction with their plant hosts. However, the corresponding signaling cascades remain largely unknown. Here, we advance our understanding of the largely unknown mechanisms by which IAA carries out its regulatory functions in plant-associated bacteria. We showed that IAA caused important changes in the global transcriptome of the rhizobacterium
IMPORTANCE
Signal sensing plays an important role in bacterial adaptation to ecological niches and hosts. This communication appears to be particularly important in plant-associated bacteria since they possess a large number of signal transduction systems that respond to a wide diversity of chemical, physical, and biological stimuli. IAA is emerging as a key inter- and intra-kingdom signal molecule that regulates a variety of bacterial processes. However, despite the extensive knowledge of the IAA-mediated regulatory mechanisms in plants, IAA signaling in bacteria remains largely unknown. Here, we provide insight into the diversity of mechanisms by which IAA regulates primary and secondary metabolism, biofilm formation, motility, antibiotic susceptibility, and phage sensitivity in a biocontrol rhizobacterium. This work has important implications for our understanding of bacterial ecology in plant environments and for the biotechnological and clinical applications of IAA, as well as related molecules.
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