Critical Reviews in Microbiology

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Activity, distribution and function of indole-3-

acetic acid biosynthetic pathways in bacteria

Cheryl L. Patten, Andrew J. C. Blakney & Thomas J. D. Coulson

To cite this article: Cheryl L. Patten, Andrew J. C. Blakney & Thomas J. D. Coulson (2013)
Activity, distribution and function of indole-3-acetic acid biosynthetic pathways in bacteria, Critical
Reviews in Microbiology, 39:4, 395-415, DOI: 10.3109/1040841X.2012.716819

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Critical Reviews in Microbiology, 2013; 39(4): 395–415
© 2013 Informa Healthcare USA, Inc.
ISSN 1040-841X print/ISSN 1549-7828 online
DOI: 10.3109/1040841X.2012.716819

Review Article

 ctivity, distribution and function of indole-3-acetic acid

A
biosynthetic pathways in bacteria
Cheryl L. Patten, Andrew J. C. Blakney, and Thomas J. D. Coulson

Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada

Abstract
The capacity to produce the phytohormone indole-3-acetic acid (IAA) is widespread among bacteria that inhabit
diverse environments such as soils, fresh and marine waters, and plant and animal hosts. Three major pathways for
bacterial IAA synthesis have been characterized that remove the amino and carboxyl groups from the 𝛼-carbon of
tryptophan via the intermediates indolepyruvate, indoleacetamide, or indoleacetonitrile; the oxidized end product
IAA is typically secreted. The enzymes in these pathways often catabolize a broad range of substrates including
aromatic amino acids and in some cases the branched chain amino acids. Moreover, expression of some of the genes
encoding key IAA biosynthetic enzymes is induced by all three aromatic amino acids. The broad distribution and
substrate specificity of the enzymes suggests a role for these pathways beyond plant-microbe interactions in which
bacterial IAA has been best studied.
Keywords: Indolepyruvic acid pathway, indoleacetamide pathway, indoleacetonitrile pathway, phylogenetics of
IAA genes, IAA gene regulation

Introduction
gall disease by the actinomycete Rhodococcus fascians
The plant hormone indole-3-acetic acid (IAA) is well (Vandeputte et al., 2005). Paradoxically, promotion of
known for its role in plant growth and development plant root development by soil bacteria Azospirillum
including apical dominance, vascular tissue diffe­ brasilense and Enterobacter cloacae (previously
rentiation, initiation of lateral and adventitious roots, misidentified as Pseudomonas putida) is also
elongation growth in stems and roots, cell division, dependent in part on IAA production by those strains
and tropic responses to gravity and light (recently (Dobbelaere et al., 1999; Patten and Glick, 2002b).
reviewed by Peer et al., 2011, and Sánchez-Rodríguez Many other plant growth-promoting rhizobacteria
et al., 2010). The widespread production of IAA among including several species of Azotobacter, Pseudomonas,
soil bacteria has received much attention as a mechanism and Bacillus (Ahmad et al., 2008; Raddidi et al., 2008;
by which plant-associated bacteria influence host plant Hassen and Labuschagne, 2010; Sokolova et al., 2011),
health. Early work with Agrobacterium tumefaciens and various Rhizobia (Vega-Hernández et al., 2002; Theunis
Pseudomonas syringae pv. savastanoi characterized et al., 2004), Methylobacterium extorquens (Ivanova et
bacterial IAA as a virulence factor that induced gall al., 2001), and Paenibacillus polymyxa (Lebuhn et al.,
tumors (Smidt and Kosuge, 1978; Thomashow et al., 1997), have been shown to produce IAA, although the
1984). Since then, bacterial IAA has been implicated importance of IAA in the beneficial interaction between
in other infectious diseases of plants such as soft rot, these strains and their host plants remains to be
wilt, and blight diseases caused by Dickeya dadantii demonstrated empirically. Bacteria isolated from lichen
(also known as Erwinia chrysanthemi) (Yang et al., (Stenotrophomonas maltophilia, Serratia marcesens,
2007), gall disease by Pantoea agglomerans (formerly Pseudomonas spp., Acinetobacter calcoaceticus, and
Erwinia herbicola) (Chalupowicz et al., 2009), and leafy Pantoea sp.) (Liba et al., 2006), algae (Bacillus pumilus,

Address for Correspondence: Cheryl L. Patten, Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada.
E-mail: [email protected]
(Received 09 April 2012; revised 24 July 2012; accepted 26 July 2012)

395
396 C. L. Patten et al.
Bacillus licheniformis and Exiguobacterium homiense) IAA biosynthetic enzymes
(Singh et al., 2011), and mycorrhizal fungi (Pseudomonas
fluorescens) (Gamalero et al., 2003) have been shown In most known cases, bacterial IAA is synthesized from
to produce IAA in laboratory cultures; however, the tryptophan by one or more pathways (Figure 1), although
relevance of IAA produced by these bacteria in the host tryptophan-independent pathways may exist (Prinsen
environment is not known. et al. 1993). However, production of the substrate tryp-
In aquatic environments, IAA produced by Vibrio spp. tophan is energetically costly, requiring 75 high-energy
isolated from the roots of salt marsh grasses (Gutierrez phosphate bonds to synthesize one molecule, the highest
et al., 2009) and by 74% of bacteria isolated from a metabolic cost of any amino acid (Akashi and Gojobori,
freshwater marsh grass including the genera Bacillus, 2002). Therefore, high levels of IAA, which is a secreted
Enterobacter, Burkholderia, Pseudomonas, Pantoea, and tryptophan metabolite, are generally produced only
Aeromonas (Halda-Alija, 2003) may affect semi-aquatic when excess tryptophan is available. Most bacterial
plants in a manner similar to terrestrial plant-bacterial cells can synthesize tryptophan but IAA synthesis is low
interactions. However, the capacity to produce IAA is not unless tryptophan is supplied exogenously in the culture
limited to bacteria associated with terrestrial or aquatic medium or external environment (Halda-Alija, 2003;
plants. In the study of wetland sediments by (Halda-Alija, Ahmad et al., 2008; Ryu and Patten, 2008). Low levels
2003), 80% of the bacteria isolated from unvegetated sed- of IAA may be produced from tryptophan synthesized
iments were also capable of producing IAA in cultures de novo, although this must not compromise protein
supplemented with tryptophan. Many of these were the synthesis, or from tryptophan released by protein
same species as those isolated from marsh grass roots degradation.
indicating that IAA producers are not restricted to a plant Tryptophan is imported into the cytoplasm by
environment. A thermoacidophilic archaeon Sulfolobus tryptophan-specific and general aromatic amino acid
sp. isolated from a hot spring also produced IAA (Wakagi transporters (Yanofsky et al., 1991) and the amino and
et al., 2002). carboxylic acid groups are enzymatically removed from
IAA has been detected in animal feces (likely of both the 𝛼-carbon of the amino acid (Figure 1) before the car-
plant and bacterial origin) and is produced in vitro by bon skeleton is excreted as IAA. Some other amino acids
bacteria that infect animal hosts including Bacteroides are catabolized by similar processes, e.g. branched chain
thetaiotaomicron, an abundant inhabitant of the human and other aromatic amino acids, although in many cases
intestine (Chung et al., 1975; Yokoyama and Carlson, the carboxylate intermediates are further oxidized via
1979; Smith and Macfarlane, 1996), and human patho- the tricarboxylic acid cycle (Fernández and Zuniga, 2006;
gens Clostridium difficile, Salmonella typhimurium and Teufel et al., 2010). Tryptophan catabolism to IAA in bac-
Mycobacterium tuberculosis (Elsden et al., 1976; Smith teria is achieved by three major pathways that employ
and Macfarlane, 1996; Werther et al., 2008). As a secreted, different enzymes and co-factors (Figure 1). Attempts
lipophilic weak acid, bacterial IAA can enter and influ- to isolate mutants that are completely deficient in IAA
ence the activities of host cells, especially at low pH, synthesis have for the most part failed, suggesting that a
and has been shown to have a cytotoxic effect on some bacterium often has multiple routes to IAA production
mammalian cells (de Melo et al., 2004; Ester et al., 2009). (Kuo and Kosuge, 1970; Liu et al., 1982; Abdel-Salam and
The rumen microflora of goats, sheep and cows contain Klingmüller, 1987; Manulis et al., 1991; Clark et al., 1993).
bacteria capable of producing IAA (Mohammed et al.,
2003; Attwood et al., 2006). Conversion of IAA to skatole Indoleacetamide pathway
(3-methyl-indole) by ruminal clostridia contributes to The indole-3-acetamide pathway has been principally
the odor of manure and to off-flavors of meat (Whitehead characterized in phytopathogens that induce gall tumors
et al., 2008). on host plants. The gall-forming pathogens P. syringae
Despite the detection of IAA in the cultures of many pv. savastanoi and P. agglomerans pv. gypsophiliae utilize
different bacteria and the potential impact of bacterial the indoleacetamide pathway as a virulence mechanism
IAA biosynthesis on other organisms, the pathway for during infection of olive trees and gypsophila, respec-
IAA production has not been identified for most bacteria, tively (Smidt and Kosuge, 1978; Comai and Kosuge, 1980;
although several different bacterial biosynthetic pathways Manulis et al., 1998), and Agrobacterium tumefaciens,
are known. The production of IAA by several different another commercially important phytopathogen, stimu-
pathways, often by multiple pathways in a single bacte- lates plant cell proliferation by transferring genes of the
rium, and the widespread production of IAA among pro- indoleacetamide pathway into the chromosome of a host
karyotes that inhabit diverse environments suggest that cell (Thomashow et al., 1984).
IAA has an important role in bacterial physiology beyond The indoleacetamide pathway converts trypto-
plant interactions. This review examines the biochemistry phan to IAA in two steps. In the first step, the enzyme
and genetics of bacterial IAA production with the aim of L-tryptophan 2-monooxygenase catalyzes the oxidative
furthering our understanding of the distribution of IAA decarboxylation of L-tryptophan to the amide indole-
biosynthetic genes among bacteria and the roles of the 3-acetamide (Figure 1). Indoleacetamide is subse-
enzymes in the physiology and ecology of the bacterium. quently deaminated oxidatively by indole-3-acetamide

 Critical Reviews in Microbiology

 Indole-3-acetic acid biosynthetic pathways 397

Figure 1. Three major IAA biosynthetic pathways in bacteria. Precursors, intermediates, and products are shown in the dominant ionization
state at physiological pH. Broken arrows indicate reactions for which the enzymes have not yet been identified in bacteria. AAT, aromatic
aminotransferase; FAD, coenzyme flavin adenine dinucleotide; GLU, glutamate; IAH, indoleacetamide hydrolase; IPDC, indolepyruvate
decarboxylase; KG, amino acceptor α-ketoglutarate; NIT, indoleacetonitrilase; NHase, indoleacetonitrile hydratase; Oxd, indoleacetaldoxime
dehydratase; PLP, coenzyme pyridoxal 5’-phosphate; TMO, tryptophan 2-monooxygenase; TPP, coenzyme thiamine pyrophosphate.

hydrolase yielding IAA and ammonia, which provides effect on the action of tryptophan 2-monooxygenase,
assimilable nitrogen. In the decarboxylation of trypto- with a Ki of 150 µM (Emanuele et al., 1995).
phan, tryptophan 2-monooxygenase with flavin adenine Tryptophan 2-monooxygenase with L-lysine 2-mono-
dinucleotide as a cofactor uses molecular oxygen as oxygenase and L-phenylalanine oxidase are different
the oxidant (Emanuele and Fitzpatrick, 1995b). Early from other members of the flavin-dependent L-amino
work with tryptophan 2-monooxygenase purified from acid oxidase family in that they catalyze the decar-
P. syringae pv. savastanoi showed that the enzyme boxylation of their amino acid substrates rather than
actually has a relatively low affinity for L-tryptophan deamination (Flashner and Massey, 1974; Koyama, 1982;
(Km = 50 µM) (Hutcheson and Kosuge, 1985). This ensures Emanuele et al., 1995; Fitzpatrick, 2010). Phenylalanine
that IAA is produced only when tryptophan is available oxidase purified from Pseudomonas sp. P-501 catalyzed
in excess of normal cellular levels (~20 µM) and there- both oxidative decarboxylation and oxidative deamina-
fore does not interfere with utilization of tryptophan for tion of phenylalanine in the same oxygen consuming
protein synthesis. At the same time, tryptophan 2-mono- reaction, producing phenylacetamide and phenylpyru-
oxygenase is negatively regulated by its reaction products vate, respectively (molar ratio 4:1) (Koyama, 1982). In
indoleacetamide and IAA at rates of 50% inhibition at 25 contrast, the activity of the tryptophan monooxygenase
µM and 230 µM, respectively. Although tryptophan is from P. syringae pv. savastanoi was restricted to produc-
the primary substrate for tryptophan 2-monooxygenase, tion of the amide (Emanuele and Fitzpatrick, 1995a,
catabolism of phenylalanine to phenylacetamide has 1995b). An amino acid oxidase from the plant pathogen
also been reported, albeit the enzyme has a much lower Ralstonia solanacearum was found to catalyze both
affinity for L-phenylalanine (Km = 2.4 mM) (Emanuele oxidative deamination and decarboxylation of all three
et al., 1995). Phenylacetamide also has a slight inhibitory aromatic amino acids (Kurosawa et al., 2009). More

© 2013 Informa Healthcare USA, Inc.

398 C. L. Patten et al.
indoleacetamide than indolepyruvate was produced of 60 kDa subunits that form a hydrophobic pocket to
from tryptophan. Interestingly, the enzyme was active accommodate the large indole ring of indolepyruvate
only after proteolytic cleavage (Kurosawa et al., 2009). (Schütz et al., 2003a). Each subunit binds one molecule
Following oxidative decarboxylation of tryptophan, of thiamine diphosphate and one molecule of Mg2+ that
the resulting indole-3-acetamide is hydrolyzed to IAA are required for tetramerization, substrate binding, and
and ammonia by the enzyme indole-3-acetamide hydro- catalytic activity (Koga et al., 1992; Schütz et al., 2003a;
lase. Indoleacetamide hydrolase is a member of the Schütz et al., 2005). Decarboxylation results from bind-
nitrilase superfamily of enzymes that hydrolyze carbon- ing of thiamine diphosphate to the keto-carbonyl group
nitrogen bonds in amides (R-C=O(NH2)) and nitriles of indolepyruvate and, following protonation of the
(R-C≡N) (reviewed by Pace and Brenner, 2001). All activated intermediate, indoleacetaldehyde is released
members of this large family possess the catalytic triad (Schütz et al., 2003a; Pohl et al., 2004). Other thiamine
glutamate-lysine-cysteine and generally exhibit broad- diphosphate-dependent 𝛼-keto acid decarboxylases,
substrate specificity. Indoleacetamide hydrolase from including phenylpyruvate decarboxylase and pyruvate
A. tumefaciens has a high affinity for indoleacetamide decarboxylase, utilize a similar catalytic mechanism
(Km = 1.2 µM), and therefore this is likely the natural sub- (Pohl et al., 2004).
strate for the enzyme in vivo; however, the enzyme is also The role of indolepyruvate decarboxylase encoded
capable of hydrolyzing several other substrates, includ- by the ipdC gene in IAA production has been experi-
ing phenylacetamide and indole-3-acetonitrile to phen- mentally confirmed in E. cloacae (Koga et al., 1992; Ryu
ylacetic acid and IAA, respectively (Kemper et al., 1985). and Patten, 2008), A. brasilense (Costacurta et al., 1994;
Malhotra and Srivastava, 2008) and P. agglomerans
Indolepyruvate pathway (Brandl and Lindow, 1996). Conversion of indolepyru-
A second major route to IAA synthesis is via the interme- vate to indoleacetaldehyde by indolepyruvate decar-
diate indole-3-pyruvate. The first step in the indolepy- boxylase is the rate-limiting step in this pathway to IAA
ruvate pathway is the transfer of the amino group from synthesis. In E. cloacae, L-tryptophan aminotransferase
tryptophan to 𝛼-ketoglutarate by an aminotransferase has a higher affinity for indolepyruvate (Km = 24 µM)
yielding indole-3-pyruvate and L-glutamate (Figure 1). than for tryptophan (Km = 3.3 mM); however, when ipdC
This reaction can be carried out by general or tryptophan- is expressed, indolepyruvate decarboxylase, which has a
specific aminotransferases that transfer the amino group high affinity for indolepyruvate (Km = 15–20 µM) (Koga
from the 𝛼-carbon of tryptophan to 𝛼-ketoglutarate. et al., 1994; Schütz et al., 2003b), drives the reaction for-
These enzymes have been extensively characterized in ward to produce IAA.
lactic acid bacteria used in the dairy industry as degrada- The A. brasilense enzyme was originally characterized
tion of aromatic and other amino acids via 𝛼-keto acid as indolepyruvate decarboxylase (Costacurta et al., 1994)
intermediates contributes characteristic flavors to vari- but has been re-designated as a phenylpyruvate decar-
ous cheeses (Helinck et al., 2004; Ardö, 2006). In many boxylase based on its greater activity with phenylpyru-
lactic acid bacteria, aromatic amino acid catabolism vate as a substrate than with indolepyruvate (Spaepen
is initiated by aminotransferases with broad substrate et al., 2007). Although the enzyme has a higher affinity
specificity that can utilize all three aromatic amino acids for indolepyruvate (Km = 0.13 mM) than for phenylpyru-
as well as methionine and leucine (Yvon et al., 1997; vate (Km = 1.08 mM), the catalytic rate is almost 100-fold
Rijnen et al., 1999; Fernández and Zuniga, 2006; Liu higher with phenylpyruvate as substrate (Spaepen et al.,
et al., 2008). Often several aromatic aminotransferases 2007). Furthermore, the A. brasilense enzyme possesses a
are present in a cell (Kittell et al., 1989). The tryptophan leucine in place of a conserved glutamate in the catalyti-
aminotransferase purified from E. cloacae exhibited a cally important amino acids found in other 𝛼-keto acid
low affinity for tryptophan (Km = 3.3 mM) (Koga et al., decarboxylases (Asp-His-Glu catalytic triad), which may
1994), and because endogenous levels of free tryptophan account in part for the preference for phenylpyruvate
are typically much lower (~20 µM), this may explain the (Versées et al., 2007), although experimental evidence
requirement for an exogenous supply of tryptophan for for this is not yet available for the A. brasilense enzyme.
IAA synthesis by this pathway. However, the S. cerevisiae phenylpyruvate decarboxylase
The second step in the indolepyruvate pathway is possesses a glutamate in the corresponding position and
the non-oxidative decarboxylation of indole-3-pyruvate replacing this with a leucine increased the affinity of the
to indole-3-acetaldehyde by indolepyruvate decar- enzyme for both phenylpyruvate and indolepyruvate,
boxylase. This enzyme belongs to a family of thiamine contrary to expectation (Kneen et al., 2011). In contrast to
diphosphate-dependent decarboxylases whose catalytic the enzyme from A. brasilense, phenylpyruvate was not a
capabilities include amino acid synthesis (e.g. acetolac- substrate for the indolepyruvate decarboxylase purified
tate synthases) or degradation (indolepyruvate and phe- from E. cloacae FERM BP-1529 (Koga et al., 1992).
nylpyruvate decarboxylases), or production of alcohols In general, the 𝛼-keto acid decarboxylases have broad
(e.g. pyruvate decarboxylases). As for other thiamine substrate specificity. While the indolepyruvate decarbox-
diphosphate-dependent decarboxylases, indolepyru- ylase from E. cloacae has high affinity for and catalytic
vate decarboxylase is a homotetramer and is made up efficiency with indolepyruvate, it can also decarboxylate

 Critical Reviews in Microbiology

 Indole-3-acetic acid biosynthetic pathways 399
pyruvate and benzoylformate, albeit with reduced the Ehrlich pathway of yeast (Hazelwood et al., 2008).
efficiency (Schütz et al., 2003b). Similarly, the pyru- Interestingly, the thermoacidophilic archaeon, Sulfolobus
vate decarboxylase from Saccharomyces cerevisiae can sp. posseses a bifunctional enzyme that catalyzes both the
decarboxylate both pyruvate and indolepyruvate but the decarboxylation of indolepyruvate and the oxidation of
same enzyme from the bacterium Zymomonas mobilis, indoleacetaldehyde (Wakagi et al., 2002).
which is similar to those from plants, cannot (Schütz et
al., 2003b). Bulky amino acid residues in the Z. mobilis Indoleacetonitrile pathway
pyruvate decarboxylase substrate binding site reduce IAA synthesis via the aldoxime-nitrile pathway has not
the volume of the cavity such that it cannot accommo- been well studied in bacteria, although some of the par-
date the larger substrate (Schütz et al., 2003a). This sug- ticipating enzymes have been identified. In this pathway,
gests that substitution of only a few amino acids in the metabolism of tryptophan to IAA proceeds through the
active site can convert a pyruvate-specific decarboxylase intermediates indole-3-acetaldoxime and indole-3-ace-
to one that can catabolize indolepyruvate in the IAA tonitrile (Figure 1). The enzymes that catalyze the synthe-
biosynthesis pathway. sis of indoleacetaldoxime have not been characterized in
Phi et al. (2008) determined that an indolepyru- bacteria; however, a cytochrome P450 monooxygenase
vate decarboxylase homologue in the genome of catalyzes this reaction in Arabidopsis (Mikkelsen et al.,
Paenibacillus polymyxa E681 was capable of producing 2000). The aldoxime group (-CH=NOH) is then dehy-
indoleacetaldehyde from indolepyruvate. Although the drated to a nitrile (-C≡N) by an aldoxime dehydratase
substrate specificity of this enzyme was not reported, yielding indole-3-acetonitrile. An aldoxime dehydratase
the amino acid sequence shows that the protein lacks from Bacillus sp. OxB-1 has been purified and character-
the conserved amino acid residues for binding indolepy- ized as a heme-containing phenylacetaldoxime dehy-
ruvate that are invariant among known indolepyruvate dratase with a requirement for flavin mononucleotide
decarboxylases (Schütz et al., 2003a; Phi et al., 2008). (Kato et al., 2000; Asano and Kato, 1998). Although the
Rather, this protein bears resemblance to the large sub- enzyme exhibited the highest affinity for and catalytic
unit of acetolactate synthase, a thiamine diphosphate- activity with phenylacetaldoxime as a substrate, it was
dependant decarboxylase that catalyzes the first step in also able to catabolize indoleacetaldoxime and several
the synthesis of branched-chain amino acids (leucine, other arylacetaldoxime substrates to corresponding
isoleucine, and valine) from two molecules of pyruvate. arylacetonitriles (Kato et al., 2000). Similarly, an aldox-
The encoding gene is found upstream of a putative aceto- ime dehydratase purified from Rhodococcus globerulus
lactate synthase small subunit gene in the P. polymyxa utilized both phenyl- and indole-acetaldoxime as sub-
E681 genome. Thus, the primary function of this enzyme strates although the preferred substrates appeared to be
may not be the production of IAA, but it may incidentally alkylaldoximes (Xie et al., 2003).
decarboxylate indolepyruvate. Organic nitriles such as indoleacetonitrile can be
Other thiamine diphosphate-dependant decarbox- converted to their corresponding carboxylic acids either
ylases can utilize both aromatic and branched-chain directly by a nitrilase or via a two-step pathway in which
𝛼-keto acids as substrates as exemplified by 𝛼-keto acid the nitrile is first hydrated to an amide by a nitrile hydra-
decarboxylases from Lactococcus lactis (Smit et al., tase and then to the carboxylic acid via an amidase.
2005) and Mycobacterium tuberculosis (Werther et al., Nitrilases belong to a family of enzymes that contain the
2008). The decarboxylase from L. lactis (KdcA) catalyzed catalytic triad glutamate-lysine-cysteine and catalyze the
the decarboxylation of the 𝛼-keto acids derived from hydration of carbon-nitrogen triple bonds to produce car-
leucine, isoleucine, valine, phenylalanine and trypto- boxylic acids and ammonia. Nitrilases generally exhibit
phan to produce aldehydes that contribute to the flavor broad substrate specificity with most showing highest
of cheese (Smit et al., 2005). The decarboxylase from activity with aromatic nitriles (O’Reilly and Turner, 2003).
M. tuberculosis (MtKDC) showed highest cata- A nitrilase purified from the soil bacterium P. fluorescens
lytic efficiency with substrates indolepyruvate and DSM 7155 showed a preference for arylacetonitriles,
𝛼-ketoisocaproate, derived from deamination of tryp- especially phenylacetonitrile; indoleacetonitrile was
tophan and leucine, respectively, but was also able to not among the substrates tested (Layh et al., 1998). This
catabolize the 𝛼-keto acid derivatives of phenylalanine, bacterium was able to utilize phenylacetonitrile as a sole
valine and isoleucine (Werther et al., 2008). source of nitrogen, and the presence of ammonia in the
The final step in the indolepyruvate pathway is the oxi- culture medium repressed the activity of the nitrilase
dation of indoleacetaldehyde to IAA. Almost nothing is (Layh et al., 1998). A small amount of the correspond-
known of the bacterial oxidases that catalyze this reaction. ing amide was also produced during the reaction as has
An indoleacetaldehyde oxidase with oxygen as the elec- been demonstrated for some other nitrilases (O’Reilly
tron acceptor may catalyze this reaction yielding IAA and and Turner, 2003; Layh et al., 1998). The phytopathogen
hydrogen peroxide as it does in some plants (Koshiba et al., P. syringae B728a was able to use indoleacetonitrile as
1996; Sekimoto et al., 1998; Seo et al., 1998). Alternatively, a source of nitrogen for growth and to produce IAA in
an aldehyde dehydrogenase with NAD+ or NADP+ as media supplemented with indoleacetonitrile whereas
the electron acceptor may catalyze this step as it does in a mutant carrying an insertion in a gene with sequence

© 2013 Informa Healthcare USA, Inc.

400 C. L. Patten et al.
homology to the Bacillus sp. OxB-1 nitrilase gene was sequences supports the distinction of these two groups of
deficient in both activities (Howden et al., 2009). A gene proteins which share <30% identity (Figure 2A). Whereas
downstream of the P. syringae B728a nitrilase gene is pre- the active sites of the indolepyruvate and pyruvate decar-
dicted to encode an acetaldoxime dehydratase (Howden boxylases carry the conserved catalytic triad Asp29-
et al., 2009), an arrangement similar to that found in His115-Glu468 that is important for substrate binding
Bacillus sp. OxB-1 (Feil et al., 2005), although the cata- and decarboxylation (Schütz et al., 2003a; Schütz et al.,
bolic activity of the protein has not been demonstrated. 2005), a leucine is found in the site corresponding to
Indoleacetonitrile can also be converted to IAA via Glu468 in the phenylpyruvate decarboxylases (Figure 2B)
indoleacetamide. Nitrile hydratases that catalyze the (Spaepen et al., 2007; Versées et al., 2007). More distantly
synthesis of amides from corresponding nitriles are not related homologues found in many other bacteria cluster
members of the nitrilase superfamily; however, amidases with known acetolactate synthases (Figure 2A).
do belong to this group of C-N hydrolyzing enzymes In contrast to ipdC of the indolepyruvic acid pathway
(Pace and Brenner, 2001). Indoleacetonitrile hydratase for IAA synthesis, homologues of the iaaM gene encod-
activity has been detected in Agrobacterium, Rhizobium, ing tryptophan 2-monoxygenase, a key enzyme in the
and Rhodococcus (Kobayashi et al., 1995; Xie et al., 2003). indoleacetamide pathway, are present in relatively few
The indoleacetonitrile hydratase purified from A. tumefa- bacterial genera (Figure 3). Phylogenetic analysis of
ciens IAM B-261 showed high catalytic activity with both translated iaaM-like bacterial sequences revealed two
indoleacetonitrile and phenylacetonitrile (Kobayashi clusters of genes (Figure 3). Group I genes include those
et al., 1995). that have been experimentally confirmed to encode
tryptophan 2-monooxygenases in the plant pathogens
A. tumefaciens, P. syringae pv. savastanoi, P. agglomerans,
Phylogenetics of IAA biosynthesis genes
and D. dadantii (Comai and Kosuge, 1982; Thomashow
Genes for IAA biosynthesis are present in a wide vari- et al., 1984; Van Onckelen et al., 1986; Clark et al., 1993;
ety of organisms, both prokaryotes and eukaryotes. Yang et al., 2007). This group is also comprised of homol-
While many of the genes for IAA synthesis in plants ogous sequences from Burkholderia spp., Agrobacterium
have remained elusive, they are better characterized in spp. and a few other P. syringae strains, many of which are
bacteria. Homologues of indolepyruvate decarboxylase known plant pathogens (Figure 3). Notably, among the
encoded by the ipdC gene are present in many diverse available P. syringae genome sequences only P. syringae
groups of bacteria that are common inhabitants of soils pv. savastanoi, pv. syringae B728a, pv. syringae Y30,
and plant and animal hosts (Figure 2A); however, the pv. glycinea, and pv. aceris have a group I sequence.
function of this gene in IAA biosynthesis has been con- These P. syringae sequences share >90% sequence iden-
firmed by mutagenesis in only a few bacteria including tity. In Agrobacterium spp., P. syringae pv. savastanoi,
E. cloacae, A. brasilense, and P. agglomerans (Koga et al., P. agglomerans, Burkholderia phymatum STM815, and
1991; Costacurta et al., 1994; Brandl and Lindow, 1996; Burkholderia sp. CCGE1002, the iaaM genes are car-
Malhotra and Srivastava, 2008; Ryu and Patten, 2008). ried on plasmids (Comai and Kosuge, 1982; Thomashow
Sequences with high similarity to the well character- et al., 1984; Barash and Manulis-Sasson, 2009).
ized ipdC gene in E. cloacae (Ryu and Patten, 2008; Koga Interestingly, while the genomes from most
et al., 1991) are present in several genera of enterobacte- sequenced P. syringae pathovars do not contain a group I
ria including Salmonella, Serratia, Klebsiella, Citrobacter, iaaM sequence, P. syringae pv. syringae B728a possesses
Yersinia, Pantoea, and Erwinia but not E. coli, Shigella, two iaaM-like genes (Figure 3). In addition to the group
or Dickeya species, and in actinobacteria Mycobacterium I protein, P. syringae B728a has a sequence that clusters
and Corynebacterium (Figure 2A). The 𝛼-keto acid with the group II sequences; the two proteins share only
decarboxylases from firmicutes Staphyloccocus, ~30% identity. Group II iaaM homologues are present
Lactococcus, Clostridium, and Bacillus are closely related in a greater diversity of bacteria including several pro-
to the indolepyruvate decarboxylases (Figure 2A). The teobacteria and actinobacteria (Figure 3). Many of these
weak statistical support for the branch separating the are common soil inhabitants. The function of the group
enteric/actinobacterial indolepyruvate decarboxylase II homologues in IAA biosynthesis has not been shown
sequences from the firmicute 𝛼-keto acid decarboxylases experimentally in any of these strains. On the other hand,
sequences may reflect an inability to clearly separate among the group II sequences is one from P. putida
these sequences into two groups. This is supported by the KT2440 (DavB) that has been determined experimentally
overlap in enzymatic activity; some enzymes from both to have lysine monooxygenase activity (Revelles et al.,
groups can catalyze the decarboxylation of 𝛼-keto acids 2005). It was not determined whether tryptophan is also
derived from branched-chain and aromatic amino acids. a substrate for this enzyme although a mutant strain was
Biochemical characterization of the homologue in able to grow on tryptophan but not lysine as a nitrogen
A. brasilense revealed that phenylpyruvate is the pre- source (Revelles et al., 2005). The P. putida lysine mono-
ferred substrate leading to redesignation of the gene as oxygenase shares a high degree of similarity (>90%) with
ppdC (Spaepen et al., 2007). Phylogenetic analysis of sequences from the other Pseudomonas spp. in this clus-
the amino acid sequences encoded by ipdC and ppdC ter. The iaaM homologues in this group may function in

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 Indole-3-acetic acid biosynthetic pathways 401

Figure 2. Phylogenetic tree of bacterial indolepyruvate decarboxylase homologues (A). Labeled groups are distinguished based on
similarity to sequences of known function (highlighted in bold) and among the sequences in the group (ALS, acetolactate synthase; IPDC,
indolepyruvate decarboxylase; KDC, 𝛼-keto acid decarboxylase; PDC, pyruvate decarboxylase; PPDC, phenylpyruvate decarboxylase;).
Known indolepyruvate decarboxylase sequences from E. cloacae UW5 and A. brasilense Sp245 were used as queries to identify homologous
sequences in GenBank (Benson et al., 2008) using BLAST (Altschul et al., 1997). A multiple sequence alignment of the protein sequences (481
amino acids) was assembled using MEGA 5 (Tamura et al., 2011) and maximum likelihood phylogenetic trees were estimated using PhyML
3.0 (Guindon et al., 2005). The phylogeny was statistically analyzed with the aLRT (Anisimova and Gascuel, 2006) under the WAG substitution
model (aLRT values are shown at nodes). GenBank accession numbers are shown. (B) Multiple alignment of amino acids of the Asp29-
His115-Glu468 catalytic triad (denoted by an asterisk) and flanking residues.

pathways other than IAA synthesis, although many are pv. syringae Y30 (White and Ziegler, 1991; Mazzola
annotated as putative tryptophan 2-monooxygenases and White, 1994), P. agglomerans (Clark et al., 1993)
based on sequence identity in the absence of experimen- and D. dadantii 3937 (Yang et al., 2007), tms-2 from
tal evidence. A. tumefaciens C58 (Schröder et al., 1984; Thomashow
Two sequences, YP002871789 and YP001748049 from et al., 1984) and aux1 from A. rhizogenes A4 (Camilleri
P. fluorescens SBW25 and P. putida W619, respectively, and Jouanin, 1991). A sequence from P. putida ATCC
share low identity (<27%) with those from group I or II 12633 that clusters with known indoleacetamide
(Figure 3). Although these are annotated as amine oxi- hydrolases (Figure 4) has been characterized as a
dases, their function remains unknown. mandelamide hydrolase (Gopalakrishna et al., 2004).
Genes encoding amidases that are homologues of This enzyme converts mandelacetamide to mandelate
indoleacetamide hydrolase are found in a great diversity (2-hydroxy-2-phenylacetic acid) that can be used as
of bacterial genera (Figure 4) as well as in eukaryotes a carbon source by P. putida (Fewson, 1988; McLeish
(Lehmann et al., 2010); however, only in a few cases et al., 2003; Gopalakrishna et al., 2004). Phenylacetic acid
have the encoded proteins been shown experimentally is also produced by this enzyme which demonstrates
to synthesize IAA. These include iaaH from P. syringae more than five-fold higher affinity for phenylacetamide
pv. savastanoi (Comai and Kosuge, 1983), P. syringae than for mandelacetamide (Gopalakrishna et al., 2004).

© 2013 Informa Healthcare USA, Inc.

402 C. L. Patten et al.

Figure 3. Phylogenetic tree of bacterial tryptophan 2-monooxygenase homologues. Groups I and II are distinguished based on sequence
similarity to sequences of known function (highlighted in bold) and among the sequences in the group. Known tryptophan 2-monooxygenase
sequences from P. syringae pv. savastanoi and A. tumefaciens C58 were used as queries to identify homologous sequences in GenBank
(Benson et al., 2008) using BLAST (Altschul et al., 1997). A multiple sequence alignment of the protein sequences (376 amino acids) was
assembled and maximum likelihood phylogenetic trees were estimated as described in the legend for Figure 2 (aLRT values are shown at
nodes). GenBank accession numbers are shown.

Other amidases that are closely related to known bac- the hydrolysis of phenylacetamide and some other
terial indoleacetamide hydrolases include an aspartate/ amides in addition to carbaryl (1-naphthyl methylcar-
glutamate tRNA-dependent amidotransferase (GatA) bamate), a commonly applied insectide (Hashimoto
that is found in diverse prokaryotic taxa (Sheppard et al., 2006).
and Söll, 2008), and a group that contains probable In some cases, iaaH genes are found adjacent to
5-aminovaleramide hydrolases (DavA) (Figure 4). GatA the genes for indoleacetamide synthesizing enzymes
transfers an amide group to aspartate or glutamate on (Figure 5). For example, the iaaH and iaaM genes of the
misacylated tRNAs (tRNAAsn and tRNAGln) to generate indoleacetamide pathway are transcribed together in
asparagine-tRNA and glutamine-tRNA, respectively, a bicistronic operon on the plasmids pIAA1 in P. syrin-
and is typically found in an operon with genes encod- gae pv. savastanoi (Comai and Kosuge, 1982; Comai
ing GatB and GatC (Figure 5), the other subunits of this and Kosuge, 1983) and pPATH in P. agglomerans (Clark
heterotrimeric enzyme (Curnow et al., 1997; Curnow et al., 1993), and are adjacent in the genomes of P. syringae
et al., 1998; Akochy et al., 2004). Together with lysine B728a and D. dadantii 3937, and in the T-DNA region of
monooxygenase (DavB), DavA degrades lysine to 5-ami- A. tumefaciens and A. rhizogenes plasmids (Figure 5). In
novalerate via 5-aminovaleramide (Revelles et al., 2005). pPATH from P. agglomerans pv. gypsophilae, the iaaMH
A homologue in Rhodococcus sp. (AAA26183; Figure 4) operon is encoded on a pathogenicity island with other
has been characterized as an arylpropionamide hydro- virulence genes (Barash and Manulis-Sasson, 2009).
lase, although arylacetamides such as indoleacetamide Related genes davB and davA also form an operon in P.
and phenylacetamide were not tested as substrates putida KT2440 (Revelles et al., 2005) and davA and davB
(Mayaux et al., 1991). The carbaryl hydrolase from homologues are found together in other pseudomonad
Arthrobacter sp. RC100 (Figure 4) was shown to catalyze genomes (Figure 5).

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 Indole-3-acetic acid biosynthetic pathways 403

Figure 4. Phylogenetic tree of bacterial indoleacetamide hydrolase homologues. Labeled groups are distinguished based on sequence
similarity to sequences of known function (highlighted in bold) and among the sequences in the group (DavA, 5-aminovaleramide hydrolase;
GatA, glutamyl-tRNAGln/aspartyl-tRNAAsn amidotransferase; IAH, indoleacetamide hydrolase). The known indoleacetamide hydrolase from
P. syrinage pv. savastanoi was used as a query to identify homologous sequences in GenBank (Benson et al., 2008) using BLAST (Altschul
et al., 1997). A multiple sequence alignment of the protein sequences (201 amino acids) was assembled and maximum likelihood phylogenetic
trees were estimated as described in the legend for Figure 2 (aLRT values are shown at nodes). GenBank accession numbers are shown.

In some bacteria, sequences that cluster with known A. radiobacter K84, B. japonicum USDA 110 and Ruegeria
indoleacetamide hydrolases are not found next to iaaM- pomeroya DSS-3, a gene encoding a protein that is simi-
like sequences (Figure 5). Interestingly, some iaaH-like lar to the indoleacetamide hydrolase from A. tumefaciens
genes are found adjacent to genes encoding nitrile and P. syringae is encoded upstream of a predicted nitrile
hydratase and therefore may deaminate indoleacet- hydratase regulatory subunit (Figure 5). Amidase/nitrile
amide produced from indoleacetonitrile (Figure 1). In hydratase gene clusters are common in Rhodococcus spp.

© 2013 Informa Healthcare USA, Inc.

404 C. L. Patten et al.

Figure 5. Genes flanking indoleacetamide hydrolase homologues in some bacterial genomes. Genes encoding predicted or confirmed
amidases including indoleacetamide hydrolases are shown in red; amine oxidases, blue; nitrile hydratase subunits, green; aryl acetaldoxime
dehydratases, yellow; and lysine monooxygenases, orange.

and some of these may catalyze the synthesis of IAA and/ in the genome; a similar aryl acetaldoxime dehydratase
or phenylacetic acid from arylacetonitriles via the corre- was not apparent in the region flanking the nitrilase in
sponding arylacetamide intermediate (Kobayashi et al., P. fluorescens EBC191 (Kiziak et al., 2005).
1993; O’Mahony et al., 2005; Okamoto and Eltis, 2007). To date, characterization of bacterial IAA biosynthetic
In R. globerulus, several genes in the indoleacetonitrile genes has focused on relatively few bacteria, mainly
pathway are found in the same region of the genome, i.e. gram-negative, plant-associated strains. The function
genes encoding an amidase, a nitrile hydratase, and an of homologues of these genes in IAA synthesis remains
aldoxime dehydratase (Xie et al., 2003), and predicted to be confirmed experimentally in other bacteria by
aryl acetaldoxime dehydratase and/or nitrile hydratase constructing mutants and by biochemical characteriza-
genes flank the indoleacetamide hydrolase homologues tion of enzyme activity including substrate preference.
in Rhodococcus sp., R. jostii RHA1, P. fluorescens SBW25 Finally, homologues of known IAA biosynthesis genes
and B. cenocepacia MCO-3 (Figure 5). Nitrile hydratases have not been found in some bacteria confirmed to pro-
have been detected in many different bacterial genera; duce IAA (e.g. Vibrio spp.). This suggests that novel IAA
however, their substrate specificities cannot be predicted biosynthetic enzymes and/or pathways remain to be
based on sequence similarity (Patek et al., 2009; Coffey discovered.
et al., 2010).
In P. fluorescens EBC191, a gene similar to known iaaH
Regulation of bacterial IAA biosynthesis
genes is found upstream of a nitrilase that was character-
ized as a phenylacetonitrilase (Kiziak et al., 2005). This It is widely believed that IAA is produced by bacteria in
nitrilase shares homology with a phenylacetonitrilase a variety of different natural environments; however,
from Bacillus OxB-1 and an indoleacetonitrilase that very few studies have actually measured this in situ. For
produces IAA in P. syringae B728a (Kato et al., 2000; example, IAA has been measured in animal feces, and in
Howden and Preston, 2009). In both of the latter bacteria, laboratory cultures of bacteria extracted from the feces,
the gene encoding an aryl acetaldoxime dehydratase that and it has been inferred from this that bacteria produce
catalyzes nitrile synthesis is adjacent to the nitrilase gene IAA in the intestinal tract of animals (Chung et al., 1975;

 Critical Reviews in Microbiology

 Indole-3-acetic acid biosynthetic pathways 405
Smith and Macfarlane, 1997). Production of IAA by bac- higher levels of tryptophan and other aromatic amino
teria in the rhizosphere or phyllosphere has largely been acids in serum compared to conventional mice, and
inferred by the loss of plant growth promotion or disease indolic products of tryptophan metabolism were signifi-
following treatment with IAA gene knockout mutants cantly higher in conventionally-raised mice compared to
(e.g. Smidt and Kosuge, 1978; Dobbelaere et al., 1999; germ-free mice (Wikoff et al., 2009).
Patten and Glick, 2002b), by an increase in plant IAA Tryptophan is not only a precursor for IAA synthesis
levels following treatment with IAA-producing bacte- but is known to induce expression of IAA biosynthetic
ria (Kaneshiro and Kwolek, 1985; Hunter, 1987), and by genes. The ipdC gene encoding indolepyruvate decar-
observation of effects that are analogous to the applica- boxylase is induced by tryptophan via the regulatory
tion of exogenous IAA (Thimann and Lane, 1938; Pilet protein TyrR in E. cloacae UW5 (Ryu and Patten, 2008).
and Saugy, 1987; Meuwley and Pilet, 1991; Malamy and Expression of ipdC is also increased by the other aro-
Benfey, 1997). matic amino acids phenylalanine and tyrosine in a
IAA production has been shown to be induced in TyrR-dependent manner (Ryu and Patten, 2008), which
vitro in the presence of leaf extracts from Citrus sinensis supports enzyme analyses that indicate that indolepyru-
in Xanthomonas axonopodis pv. citri (Costacurta et al., vate decarboxylase catalyzes the metabolism of a broader
1998) and extracts from leafy galls of Nicotiana taba- range of substrates in some bacteria. A TyrR box that is
cum by Rhodococcus fascians (although a supplement similar to the consensus sequence for the TyrR bind-
of tryptophan was required) (Vandeputte et al., 2005). ing site in E. coli (Pittard et al., 2005) is centered 91 bp
Moreover, ipdC promoter activity increased dramati- upstream of the start codon for ipdC in E. cloacae UW5,
cally when P. agglomerans 299R was associated with the and disruption of TyrR through insertional mutagenesis
leaves and flowers of various plants (Brandl and Lindow, resulted in lower levels of ipdC expression and loss of
1997; Brandl et al., 2001), and when A. brasilense Sp7 was IAA production (Ryu and Patten, 2008). In E. coli, TyrR
associated with wheat roots (Rothballer et al., 2005). The regulates the expression of several genes that function in
iaaM gene of D. dadantii 3937 was upregulated when aromatic amino acid transport and biosynthesis (Pittard
associated with host spinach plants (Yang et al., 2004). et al., 2005). Binding of aromatic amino acids to TyrR
While these studies suggest that IAA is produced when increases the affinity of TyrR for its recognition sequence
the bacterium is associated with a host plant, bacterial thereby increasing gene expression. A sequence similar
IAA production in situ has not been quantified. to the TyrR box consensus sequence is found upstream
Production of high levels of IAA by bacteria typically of the predicted ipdC sequences in several other enteric
requires an exogenous source of tryptophan. IAA syn- bacteria (Figure 6) suggesting that these genes are also
thesis by the indoleacetamide pathway in P. syringae pv. activated by TyrR.
syringae and D. dadantii 3937 increased upon addition of TyrR belongs to the NtrC family of transcriptional
tryptophan to the culture medium (Fett et al., 1987; Yang regulators. This family of regulators generally controls
et al., 2007) and synthesis via the indolepyruvate pathway pathways that are involved in nitrogen acquisition, and
was also induced by supplementation with tryptophan TyrR homologues have been identified in several dif-
(Kaneshiro et al., 1983; Barbieri et al., 1986; Ernstsen ferent bacteria. In Pseudomonas, the TyrR homologue
et al., 1987; Koga et al., 1991; Omay et al., 1993; Brandl PhhR binds to a consensus sequence that is identical
et al., 1996; Ona et al., 2005; Ryu and Patten, 2008). to that for TyrR in the promoter region of genes that
Although most bacteria produce tryptophan, endog- encode enzymes for catabolism of aromatic amino acids
enous levels are too low to drive production of high (Herrera et al., 2009). PhhR regulated genes in P. putida
levels of IAA (Hutcheson and Kosuge, 1985; Koga et al., KT2440 include those encoding enzymes that catabolize
1994). While tryptophan is not typically an abundant the 𝛼-keto acids derived from tyrosine and phenylala-
amino acid, it is likely to be present in the environments nine (Herrera et al., 2009). However, it remains to be seen
where IAA-producing bacteria are found. Seed and root whether PhhR controls the expression of genes required
exudates are known to contain tryptophan although
the amount of tryptophan present depends on the type,
age, and nutritional status of the plant, as well as other
environmental factors (Martens and Frankenberger,
1994; Kamilova et al., 2006). Although low the levels of
tryptophan in exudates from roots of Avena barbata were
adequate to increase the activity of a tryptophan respon-
sive promoter in P. agglomerans 299R (Jaeger et al., 1999).
Tryptophan is available to IAA-producing bacteria in the Figure 6. TyrR box sequences are conserved in the region
intestinal tract of animals from dietary proteins, and from upstream of the predicted ipdC genes in Enterobacteriaceae. The
proteins produced by intestinal bacteria and the animal Escherichia coli TyrR consensus sequence is shown. Asterisks
indicate conserved nucleotides. Ecl UW5, Enterobacter cloacae
host. Tryptophan catabolism in the intestinal tract of UW5; Sen LT2, Salmonella enterica LT2; E 638, Enterobacter sp.
mammals has been attributed to gut microorganisms 638; Pan LGM, Pantoea anantis LGM 20103; Kpn 342, Klebsiella
(Wikoff et al., 2009). Germ-free mice had significantly pneumoniae 342; Cko 895, Citrobacter kosenoi 895.

© 2013 Informa Healthcare USA, Inc.

406 C. L. Patten et al.
for IAA production in Pseudomonas spp. In Shewanella is similar to the −10 and −35 regions of the E. coli 𝜎70
spp., TyrR is predicted to control genes required for deg- consensus promoter (Gaffney et al., 1990). The level of
radation of peptides and branched chain and aromatic expression was found to be similar in the presence of
amino acids including an indolepyruvate decarboxylase several different carbon and nitrogen sources (Gaffney
homologue (Rodionov et al., 2011). et al., 1990).
In A. brasilense the ipdC promoter also responds to IAA is produced by many bacterial cultures only
exogenous tryptophan and other aromatic amino acids. when they are in the stationary phase of growth even
While expression of ipdC::gusA fusions did not increase when exogenous tryptophan is present throughout the
following addition of tryptophan in an IAA mutant growth cycle (Vande Broek et al., 2005; Ryu and Patten,
strain of A. brasilense Sp245 grown in complex medium 2008). This may be because expression of IAA genes is
(Vande Broek et al., 1999), ipdC reporter gene fusions downregulated when preferred nitrogen sources are
were induced in wild-type A. brasilense Sp245 and in A. available. Perhaps in stationary phase when more read-
brasilense Sp7 grown on minimal media supplemented ily assimilable nitrogen sources such as ammonium have
with any of the three aromatic amino acids (Ona et al., been depleted from the culture medium, nitrogen for
2005; Rothballer et al., 2005). The complex Luria-Bertani biosynthetic processes can be provided by deamination
medium contains aromatic amino acids (~1 mM tryp- of tryptophan following induction of one of the IAA bio-
tophan and tyrosine, 2.4 mM phenylalanine; (Sezonov synthetic pathways. Although there is no direct evidence
et al., 2007) that may be sufficient to activate the ipdC for repression of IAA biosynthetic genes by ammonium,
promoter in the absence of additional supplements Malhotra and Srivastava (2009) found that more IAA
(<1 mM is required for activation of the E. cloacae UW5 accumulated in cultures of A. brasilense SM grown in
ipdC promoter) (Ryu and Patten, 2008). Alternatively, medium containing 50% less ammonium sulfate. Shokri
ipdC is activated by the end product of indolepyruvate and Emtiazi (2010) also found that the source of nitro-
decarboxylase catabolism in the wild-type A. brasilense gen in the culture medium had the greatest impact on
background. In A. brasilense Sp245 and A. brasilense quantity of IAA produced by Rhizobium. Interestingly,
SM, the ipdC gene is positively controlled by IAA, and in the yeast Saccharomyces cerevisiae, which produces
the synthetic auxins 2,4-dichlorophenoxypropionic IAA via the indolepyruvic acid pathway, expression of
acid, naphthaleneacetic acid, and chlorophenoxyace- the gene encoding the broad-substrate-specificity 𝛼-keto
tic acid (Malhotra and Srivastava, 2008; Vande Broek acid decarboxylase that participates in this reaction was
et al., 1999). A sequence (TGTCCC) with homology downregulated on the preferred nitrogen source ammo-
to the cis-acting auxin-responsive elements in plants nium compared with poorer nitrogen sources such as
(TGTCNC) was identified in the A. brasilense ipdC pro- phenylalanine (Boer et al., 2007).
moter region (Lambrecht et al., 1999; Rothballer et al., Another possible explanation for the observation
2005; Malhotra and Srivastava, 2008), and point muta- that IAA is produced only in stationary phase is that
tions introduced into this sequence and an upstream IAA biosynthetic genes may be induced in response
inverted repeat sequence (ATTGTTTC-N4-GAAACAAT) to a cell-density dependent signal. In some bacteria,
abolished or reduced expression of ipdC in response to IAA production and ipdC expression are regulated by
IAA (Vande Broek et al., 2005). Auxin-responsive ele- the stationary-phase sigma factor RpoS (Patten and
ments have been found in plants upstream of genes Glick, 2002a; Saleh and Glick, 2001). Quorum-sensing
that are controlled by IAA (reviewed by Chapman and signals such as N-acylhomoserine lactones produced
Estelle, 2009). However, in plants, the transcription by gram-negative bacteria also regulate functions that
factors that bind to these promoter elements through are effective at higher cell densities including those
highly conserved DNA-binding domains do not bind to that affect host organisms such as virulence of plant
IAA directly. Rather, in the absence of auxin the tran- and animal pathogens, and biocontrol activities of
scription factors bind to the auxin-responsive elements plant growth-promoting bacteria (Waters and Bassler,
as a complex with Aux/IAA proteins and corepressor 2005). N-acylhomoserine lactones regulate bacterial
proteins thereby repressing gene transcription. When IAA production, however, they appear to have con-
present, IAA forms a complex with auxin signaling trasting effects; regulation was positive in Azospirillum
receptors and Aux/IAA which results in degradation of lipoferum B518 (Boyer et al., 2008) but negative in
Aux/IAA and derepression of promoter activity (Gray Serratia plymuthica (Liu et al., 2011; Müller et al., 2009).
et al., 2001). The proteins that mediate control of ipdC Biofilms induced by quorum-sensing signals are high-
by IAA in A. brasilense have not been identified and the density populations found on diverse biotic and abiotic
mechanism by which expression is controlled by IAA is surfaces (Hall-Stoodley et al., 2004). Microarray analysis
as yet unknown. revealed that ipdC in Salmonella typhimurium is upreg-
The genes encoding enzymes in the indoleacetamide ulated 3.5-fold in biofilm cells compared to planktonic
pathway appear to be constitutively expressed in P. syrin- cells (Hamilton et al., 2009). Interestingly, several genes
gae pv. savastanoi. Expression of the iaaMH operon in involved in tryptophan transport were also upregulated
P. syringae pv. savastanoi does not require an exog- in the biofilms and the presence of aromatic amino acids
enous supply of tryptophan and the promoter sequence (but not non-aromatic amino acids) promoted biofilm

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 Indole-3-acetic acid biosynthetic pathways 407
formation (Hamilton et al., 2009). Biofilm cells exhibit naturally secrete high levels of IAA stimulated the for-
enhanced resistant to a variety of stresses and there is mation of lateral roots on canola, Medicago, and wheat,
some evidence to suggest that IAA may be a signal that respectively (Barbieri et al., 1986; Barbieri and Galli, 1993;
mediates the stress response (see discussion below). Xie et al., 1996; Mayak et al., 1997; Bianco and Defez, 2010).
Much more needs to be done to identify the environ- An enhanced root system helps to anchor plants in soil
mental factors that increase expression of bacterial IAA and to increase the surface area through which water and
biosynthesis genes and the proteins that mediate the minerals can be obtained. On the other hand, some rhi-
response to these factors. For example, very little has zobacteria that produce high levels of IAA inhibit elonga-
been done to characterize expression of genes of the tion growth of primary roots. For example, while wild-type
indoleacetamide pathway in plant pathogens, which is P. putida GR12-2 stimulated elongation of canola seedling
surprising as IAA produced by this pathway is a virulence roots, an IAA-overproducing mutant inhibited primary
factor. Understanding how IAA production is regulated root growth (Xie et al., 1996). Similarly, an IAA overproduc-
in host organisms is especially important because both ing mutant of P. fluorescens CHA0 had a deleterious effect
pathogens and beneficial bacteria produce IAA that has on primary root growth of wheat and cucumber (Beyerler
the potential to affect host cells. et al., 1997). Decreasing the inoculum density of the
P. fluorescens mutant reversed the inhibitory effects on
Function of bacterial IAA cucumber and wheat roots (Beyerler et al., 1997). Loper
and Schroth (1986) demonstrated a linear relationship
In plant-microbe interactions between the accumulation of IAA in cultures of several
Bacterial IAA has long been known to play a role in plant rhizosphere isolates of enterobacteria and inhibition of
disease. In the phytopathogen A. tumefaciens, genes of primary root elongation in sugar beet seedlings. Early
the indoleacetamide pathway for IAA synthesis are car- work on plant responses to application of exogenous IAA
ried on the T-DNA region of the tumor-inducing (Ti) indicated that only a small window of exogenous IAA con-
plasmid that is transferred into the chromosome of centration positively influences root elongation growth
plant cells where IAA production stimulates plant cell (Thimann and Lane, 1938). The effective range varies
proliferation and crown gall tumors (Thomashow et al., according to plant species and to the stage of root devel-
1984; Van Onckelen et al., 1986; Schröder et al., 1984). opment, but is typically between 10−9 and 10–12 M (Pilet
IAA-producing strains of P. syringae and P. agglomerans and Saugy, 1987; Evans et al., 1994). Higher concentrations
pv. gypsophilae also induce gall formation on plants such inhibit root elongation likely by stimulating growth-inhib-
as olive, oleander, privet, and gypsophila although in iting levels of ethylene in plants (Peck and Kende, 1995).
these pathogens IAA biosynthetic genes are not trans- Thus, the levels of IAA secreted by a bacterium in situ may
ferred into infected host cells (Kuo and Kosuge, 1970; influence the effect on host plants.
Manulis et al., 1991; Comai et al., 1982). Abolished The IAA levels attributed to plant growth-promoting
or reduced gall formation in plants infected with rhizobacteria are determined from laboratory cultures
P. syringae and P. agglomerans mutant strains that no of these bacteria. Phenylacetic acid is typically not mea-
longer had functional IAA biosynthetic genes implicated sured in the cultures although many of these bacteria
IAA in tumorigenesis (Smidt and Kosuge, 1978; Comai utilize the indolepyruvic acid pathway to produce both
and Kosuge, 1980; Comai et al., 1982; Iacobellis et al., IAA and phenylacetic acid, and precursors of both prod-
1994; Yamada, 1993; Surico et al., 1984). IAA produced ucts induce expression of indolepyruvate decarboxylase
by the indoleacetamide pathway is also an important (Ryu and Patten, 2008). Phenylacetic acid is a weak auxin
virulence factor in nontumorigenic infectious diseases (Wightman and Lighty, 1982). Small and Morris (1990)
of plants. For example, abolition of IAA production in a observed that application of both IAA and phenylacetic
D. dadantii iaaMH mutant reduced symptoms of rot dis- acid had an additive effect on elongation of Phaseolus
ease on Saintpaulia ionantha (African violet) by decreas- vulgaris internodal segments, although phenylacetic
ing production of pectinases that degrade host cell walls acid alone stimulated a weaker response than IAA alone.
(Yang et al., 2007). Analysis of specific gene expression in On the other hand, phenylacetic acid stimulated the ini-
the D. dadantii IAA mutant suggests that disease induc- tiation of more lateral roots in pea seedlings compared to
tion by IAA likely occurs through a type III secretion IAA (Wightman et al., 1980). Undissociated phenylacetic
system and GacSA-mediated activation of extracellular acid can diffuse into plant cells and, in the neutral pH
enzymes that destroy host cells (Yang et al., 2007). environment of the cytoplasm, dissociates into anionic
Paradoxically, many plant growth-promoting bacteria phenylacetate that binds to auxin transport proteins
that inhabit the rhizosphere also produce IAA. The root- (Löbler and Klämbt, 1985). However, phenylacetate is
promoting activity of some rhizobacteria was attributed not exported by auxin efflux carriers and by inhibiting
to bacterial IAA when mutants with disruptions in IAA the activity of auxin efflux carriers may also prevent the
biosynthetic genes no longer enhanced root development polar transport of IAA (Morris and Johnson, 1987). Auxin
(Patten and Glick, 2002b; Dobbelaere et al., 1999; Barbieri gradients are considered to be important determinants
et al., 1986). IAA-overproducing mutants of P. putida and of root development, and other auxin-controlled func-
Sinorhizobium meliloti, and A. brasilense strains that tions in plants, and these in turn are influenced by auxin

© 2013 Informa Healthcare USA, Inc.

408 C. L. Patten et al.
transport (Simon and Petrášek, 2011; Dhonukshe et al., In interactions with animal cells
2008). Localized accumulation of IAA in root pericycle IAA has been shown to have a direct cytotoxic effect on
cells stimulates the formation of lateral root founder cells some mammalian cells. Neutrophils treated with IAA
that divide to form new root primordia (Dubrovsky et al., (1 mM) exhibited characteristics of apoptosis includ-
2008). Thus, the lateral root-stimulating effects of bacteria ing fragmented DNA, chromatin condensation, and
may be attributed to phenylacetate-mediated disruption depolarization of mitochondrial membranes (de Melo
of IAA transport as well as to IAA production, although et al., 2004). Apoptotic neuroepithelial cells and other
this remains to be confirmed experimentally. neurodevelopmental abnormalities were observed in rat
Auxins secreted by bacteria, whether pathogenic or and mice embryos following exposure to IAA (Furukawa
beneficial, may suppress plant defense responses to et al., 2004; Furukawa et al., 2007). IAA also inhibited the
the infecting microbe thereby promoting colonization. proliferation of several different tumor cell lines (Ester
This may occur through plant auxin signaling pathways et al., 2009). Although the mechanism by which IAA
that have been shown to influence the susceptibility affects mammalian cells is unknown, in rat liver, renal,
of plants to bacterial infection (for discussion of auxin lung, and intestinal tissues it was shown to stimulate a
signaling pathways in plant defense, please see recent two- to threefold increase in guanylate cyclase activity
review by Kazan and Manners, 2009). Downregulation and therefore production of the secondary messenger
of auxin signaling F-box receptors and auxin-responsive cGMP that regulates many important cellular processes
genes in Arabidopsis thaliana decreased infection and including apoptosis (Vesely et al., 1985). Increased gua-
disease symptoms by P. syringae DC3000 (Navarro et al., nylate cyclase activity was observed at applied IAA con-
2006). Conversely, enhanced auxin signaling and bio- centrations above 10 nM.
synthesis and exposure to exogenous auxin increased It is conceivable that IAA and phenylacetic acid pro-
colonization of P. syringae 20-fold and increased disease duced by some intestinal bacteria may influence the activ-
symptoms in infected plants (Navarro et al., 2006; Chen ity of intestinal epithelial cells. Common human intestinal
et al., 2007; Wang et al., 2007). Similarly, treatment of rice bacteria such as B. thetaiotaomicron and Clostridium spp.
with exogenous IAA promoted infection by the pathogen and enteric bacteria such as E. cloacae are capable of pro-
Xanthomonas oryzae (Ding et al., 2008). IAA biosynthe- ducing high levels of IAA and phenylacetic acid in the pres-
sis via the chromosomally encoded indolepyruvic acid ence of the aromatic amino acid precursors (Chung et al.,
pathway enhanced colonization by P. agglomerans but 1975; Elsden et al., 1976; Yokoyama and Carlson, 1979; Ryu
did not directly contribute to formation of gall tumors and Patten, 2008; Russell et al., 2011). Indeed, consump-
on gypsophila (Manulis et al., 1998). Disruption of this tion of a high protein diet was shown to raise gut phenyl-
pathway reduced the P. agglomerans population on gyp- acetic acid levels almost threefold compared with a diet
sophila and bean leaves, while disruption of the plas- that contained half as much protein (Russell et al., 2011).
mid-borne indoleacetamide pathway reduced tumor As weak acids, some of the intestinal IAA and phenylace-
formation on gypsophila by 40% (Brandl and Lindow, tic acid would be in an undissociated, lipophilic form that
1998; Manulis et al., 1998). IAA secreted by bacteria on could readily diffuse across the intestinal cell membrane.
plant surfaces may activate expansins, plant cell wall The rumen microflora of goats, sheep and dairy cows
loosening proteins, thereby weakening the protective, contain bacteria capable of producing IAA (Attwood
fibrous extracellular matrix and facilitating bacterial et al., 2006; Mohammed et al., 2003) and decarboxylation
colonization (Ding et al., 2008). of IAA to skatole (3-methyl-indole) by ruminal clostridia
Although IAA may increase plant colonization by contributes to the odor of manure and to off-flavors of
enhancing plant auxin signaling, IAA produced by rhizo- meat (Whitehead et al., 2008). More serious to livestock
bacteria may directly inhibit the activity of phytopatho- health, high levels of skatole produced from IAA fol-
gens thereby protecting plants from infectious disease. lowing ingestion of large doses of the IAA precursor
IAA (and to a lesser extent phenylacetic acid) inhibited tryptophan in feed can lead to acute pulmonary edema,
expression of virulence genes required for tumour induc- emphysema, and even death of some of the animals
tion by A. tumefaciens (Liu and Nester, 2006; Yuan et al., (reviewed by Deslandes et al., 2001). Skatole is absorbed
2008). Similarly, production of IAA by A. tumefaciens into the blood from the rumen and can destroy lung tis-
and P. syringae pv. savastanoi prevented induction of sue (Yokoyama and Carlson, 1979; Thornton-Manning
the hypersensitive response in tobacco by the pathogen et al., 1993; Linden et al., 1996). Skatole is also produced
P. syringae pv. phaseolicoli (Robinette and Matthysse, from IAA in saliva (Codipilly and Kleinberg, 2008) and in
1990). At higher concentrations IAA prevents prolifera- the human intestine, and can be adsorbed into the blood
tion of A. tumefaciens and several other beneficial and stream (Smith and Macfarlane, 1996) although its effects
pathogenic plant-associated bacteria (Liu and Nester, on human health are not known.
2006; Yuan et al., 2008). The inhibitory effect of IAA may
be attributed to its weak acid character; weak organic In bacterial physiology
acids are well known for their antimicrobial properties Although bacterial IAA plays an important role in host
(Brul and Coote, 1999). interactions, the production of IAA by many different

 Critical Reviews in Microbiology

 Indole-3-acetic acid biosynthetic pathways 409
bacteria in nonhost environments and the presence of signal. Whole genome transcriptional studies in
several IAA biosynthetic pathways, often in a single bac- E. coli, Rhizobium etli, A. tumefaciens, and A. brasilense
terial strain and often catalyzed by enzymes that are not indicated that IAA regulates bacterial gene expression
used exclusively for IAA synthesis, suggests that there are (Bianco et al., 2006a; Yuan et al., 2008; Spaepen et al.,
other important physiological roles for IAA. Catabolism 2008; Van Puyvelde et al., 2011). In R. etli, exogenous
of tryptophan, and other aromatic amino acids in some IAA increased expression of genes that may play a role in
cases, by all three major IAA biosynthetic pathways perception of and attachment to host legumes (Spaepen
releases the amine group for nitrogen metabolism. et al., 2008). Genes responsive to IAA, whether IAA is sup-
Acquisition of nitrogen by transamination of aromatic plied exogenously or produced endogenously, include
and branched-chain amino acids yields carbonyl inter- those involved in signal transduction in A. brasilense
mediates that often cannot be used as carbon sources, (Van Puyvelde et al., 2011). Many of the protein products
and are toxic when they accumulate (O’Brien et al., 2005; of IAA upregulated genes enable cells to survive a vari-
Ellis, 2007; Benigni and Bossa, 2011). Indeed, levels of the ety of stresses (Bianco et al., 2006b). For example, treat-
𝛼-keto acid indolepyruvate, and indoleacetaldehyde that ment of E. coli with 0.5 mM IAA increased expression of
is derived from it, are kept very low in the cell by conver- several genes that influence cell envelope composition,
sion to tryptophan by transaminases that have a higher and enhanced resistance to increased acidity (pH 3.0),
affinity for indolepyruvate than for tryptophan or to IAA osmolarity (0.5 M NaCl), temperature (55°C), oxidative
by indolepyruvate decarboxylase (Koga, 1995). When stress (2 mM H2O2), and antibiotics (Bianco et al., 2006b).
the concentration of tryptophan is high, production of Uptake of IAA by the recipient cell can reduce cytoplas-
IAA may provide a mechanism to detoxify these toxic mic pH and induce expression of proteins that mitigate
intermediates. stress damage. Increases in heat shock proteins, exopoly-
IAA is primarily thought of as a secreted molecule; saccharide production and biofilm formation, which are
however, some bacteria can further catabolize IAA as a known to enhance stress resistance, were observed. IAA
source of carbon for growth. Strains from the bacterial is also proposed to signal a response to environmental
genera Pseudomonas and Arthrobacter can metabolize changes in yeast (Prusty et al., 2004).
IAA via the intermediate catechol that enters the central
carbon metabolic pathway through 𝛽-ketoadipate (Mino,
Concluding remarks
1970; Gieg et al., 1996; Leveau and Lindow, 2005). Several
loci required for IAA degradation were identified in P. The capacity to synthesize IAA via the indoleacetamide
putida 1290 including an iac locus encoding proteins of pathway appears to be present in relatively few, mainly
as yet uncharacterized biochemical function and homo- phytopathogenic bacteria. In many of these bacteria,
logues of catechol-degrading enzymes (Leveau and the genes encoding tryptophan 2-monooxygenase and
Gerards, 2008). Alternatively, IAA may be degraded by a indoleacetamide hydrolase are present on a plasmid,
pathway similar to that for aerobic phenylacetate catabo- suggesting a mechanism for spread of these virulence
lism in Pseudomonas sp. Y2 (Teufel et al., 2010). In this genes. On the other hand, the production of IAA via the
pathway, a phenylacetate-CoA thioester forms and then indolepyruvate pathway appears to be more widespread.
the aromatic ring is cleaved by the introduction of oxygen. Moreover, the 𝛼-keto acid decarboxylase that catalyzes a
IAA is a membrane-permeant weak acid (pKa = 4.8) and key step in this reaction is promiscuous and can utilize
the relative levels of the protonated, lipophilic form and the 𝛼-keto acids derived from aromatic and branched-
the anionic, lipophobic form are pH dependent. At the chain amino acids as a substrate. This results in produc-
physiological pH for many of the neutralophilic bacteria tion of compounds other than IAA that may impact the
discussed above (i.e. ~pH 7.6), less than 1% of intracellu- bacterium and its hosts. For example, phenylacetic acid
lar IAA remains protonated and therefore able to diffuse as well as IAA may play a role in plant growth promotion.
across the cell membrane. The anion may be exported by Much less is known about the biochemistry and ecology
an auxin efflux carrier as in plants (recently reviewed by of the indoleacetonitrile pathway.
Petrásek and Friml, 2009) and yeast (Hazelwood et al., A major function of these pathways is the acquisition of
2006); however, such a transporter has not been identi- nitrogen through protein and amino acid catabolism. In
fied in bacteria. When the pH of the cytoplasm is reduced, Shewanella, for example, the ipdC homologue is part of a
more of the IAA produced is in the undissociated form. regulon that includes genes for degradation of peptides, and
Secretion of protonated IAA removes protons that acidify aromatic and branched-chain amino acids (Rodionov et al.,
the cytoplasm and therefore may help to maintain pH 2011). Proteins and amino acids are available for bacterial
homeostasis in bacteria. Indeed, in A. brasilense, ipdC metabolism in the intestinal tract of animals, in the rhizo-
expression was upregulated approximately threefold at sphere in plant root exudates, and in many environments
pH 5.5 compared to pH 7 and the IAA content of the cul- from lysed bacterial cells. When IAA cannot be used as a
ture medium increased 40-fold at pH 6.3 compared with carbon source it is secreted which provides a mechanism to
pH 6.8 (Vande Broek et al., 2005). prevent accumulation of toxic intermediates. As a secreted,
As a secreted product that can diffuse into neigh- lipophilic molecule, IAA has the potential to impact the
bouring cells, IAA may act as a cell–cell communication activities of other cells, whether prokaryotic, animal or plant

© 2013 Informa Healthcare USA, Inc.

410 C. L. Patten et al.
cells by altering signal transduction and regulating gene Bianco C, Imperlini E, Calogero R, Senatore B, Amoresano A, Carpentieri
expression in those cells. In environments with low pH such A, Pucci P, Defez R. (2006b). Indole-3-acetic acid improves
Escherichia coli’s defences to stress. Arch Microbiol, 185, 373–382.
as the slightly acidic rhizosphere and animal digestive tract, Bianco C, Defez R. (2010). Improvement of phosphate solubilization
diffusion of IAA into neighbouring cells would increase due and Medicago plant yield by an indole-3-acetic acid-overproducing
to increased levels of undissociated IAA and may stimulate strain of Sinorhizobium meliloti. Appl Environ Microbiol, 76,
a protective response in some bacteria. 4626–4632.
Boer VM, Tai SL, Vuralhan Z, Arifin Y, Walsh MC, Piper MD, de Winde
JH, Pronk JT, Daran JM. (2007). Transcriptional responses of
Declaration of interest Saccharomyces cerevisiae to preferred and nonpreferred nitrogen
sources in glucose-limited chemostat cultures. FEMS Yeast Res, 7,
This work was supported by grants to C.L.P. and scholar-
604–620.
ships to A.J.C.B and T.J.D.C. by the Natural Sciences and Boyer M, Bally R, Perrotto S, Chaintreuil C, Wisniewski-Dyé F. (2008).
Engineering Research Council of Canada. A quorum-quenching approach to identify quorum-sensing-
regulated functions in Azospirillum lipoferum. Res Microbiol, 159,
699–708.
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