Diversity of the Tryptophanase Gene and Its Evolutionary Implications in Living Organisms

Abstract

Tryptophanase encoded by the gene tnaA is a pyridoxal phosphate-dependent enzyme that catalyses the conversion of tryptophan to indole, which is commonly used as an intra- and interspecies signalling molecule, particularly by microbes. However, the production of indole is rare in eukaryotic organisms. A nucleotide and protein database search revealed tnaA is commonly reported in various Gram-negative bacteria, but that only a few Gram-positive bacteria and archaea possess the gene. The presence of tnaA in eukaryotes, particularly protozoans and marine organisms, demonstrates the importance of this gene in the animal kingdom. Here, we document the distribution of tnaA and its acquisition and expansion among different taxonomic groups, many of which are usually categorized as non-indole producers. This study provides an opportunity to understand the intriguing role played by tnaA, and its distribution among various types of organisms.

1. Introduction

Tryptophanase (TnaA) is a pyridoxal 5′phosphate-dependent enzyme that catalyses the hydrolytic β-elimination of tryptophan to indole, pyruvate, and ammonia, which all play unique roles within organisms and the environment [1,2,3]. Consequently, it is an important enzyme from the perspective of amino acid and nitrogen metabolism. Pyruvate is a key molecule that links carbon and nitrogen metabolic pathways, drives metabolic flux according to environmental conditions and helps to regenerate reducing equivalents under aerobic and anaerobic environments [1]. Re-routing of pyruvate toward gluconeogenesis is associated with the maintenance of enterohaemorrhagic Escherichia coli O157:H7 in bovine intestines [1]. The degradation of tryptophan, like other amino acids, results in the production of ammonia as a by-product, which in turn may participate in other biochemical reactions such as nitrogen cycle post excretion into the environment.

Indole is a unique compound with wide-ranging effects on many organisms [4,5] and its involvement as an effector molecule in prokaryotes and eukaryotes is intriguing. In prokaryotes, it serves as a signalling molecule that modulates cell division, virulence, and biofilm formation [5]. Antibiotic tolerance is a major menace in clinical medicine [6,7] and indole contributes to the resistance acting as a signalling molecule [5]. In addition, indole-producing bacteria can interfere with quorum sensing, biofilm formation, antibiotic tolerance, pigment production, and predation abilities of non-indole producing bacteria including pathogens [2,3,4,5,8,9].

In eukaryotes, indole and indole-derived compounds display a broader significance. In insects, indole is a cue to identify food, mating partners, and ovipositional sites [10,11,12,13]. Indole also participates in survival dynamics and is utilized in predator–prey interactions and plant defence mechanisms [14,15,16]. Fungi and several marine organisms can use indole to produce indole-derived compounds, such as pigments, alkaloids, Tyrian purple, and antifouling agents [17,18,19]. In vertebrates, including humans, tryptophan metabolism leads to the synthesis of indole moieties containing precursors of key mediators such as serotonin, melatonin, kynurenine, and tryptamine [20,21,22].

Thus, available evidence demonstrates that tryptophan metabolism plays vital roles in various living forms [23]. The ability of TnaA to conduct reverse α,β-elimination and β-substitution reactions has also attracted attention because the former can be used to synthesize tryptophan [24]. Furthermore, the ability of TnaA to synthesize tryptophan is important in higher eukaryotic hosts because it is used by gut microbiota to produce the rare amino acid tryptophan [1]. Therefore, we decided to study the lineage and diversity of the tnaA gene to identify the roles of indole and TnaA in organisms and biotechnological applications associated with variations in the active sites of TnaA, especially concerning the biosynthesis of hormones and alkaloids.

2. Materials and Methods

2.1. Sequence Data/Data Mining

The tnaA sequences (>1200 nts) of 221 isolates of 36 eukaryotes (Drosophila, hemichordates and marine organisms including octopus, sea anemone, corals, hermit crab), 41 archaea, 16 fungi, and 128 prokaryotes (Gram-positive and -negative bacteria) were investigated in the present study. These gene sequences were obtained from the NCBI database (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/gene accessed on 8 October 2020). The following exclusion terms were used: putative protein; TnaA leader peptide (tnaC); tryptophan 2,3-dioxygenase; and hypothetical proteins; record removed; and large eukaryote sequences (with long ‘N’ stretches). Curation of tnaA sequences was performed manually to avoid redundant/partial or non-related sequences.

2.2. Phylogenetic Analyses

For the nucleotide sequence phylogenetic tree, the 221 tnaA nucleotide sequences were assembled and translated to their respective cDNA sequences and aligned using the multiple sequence alignment program ClustalX version 2.0.12. To estimate evolutionary distances, pairwise distances between species were calculated using the MEGA 11 package. A total of 221 tnaA ortholog sequences were analysed. Sequences were aligned with MEGA 11 and alignment errors were corrected using BioEdit 7.2. Evolutionary history was inferred using the Maximum Likelihood method and General Time Reversible model. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 1.0799)). The rate variation model allowed for some sites to be evolutionarily invariable. The analysis was conducted for 1000 bootstrap replications. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 221 nucleotide sequences. There was a total of 32,115 positions in the final dataset. Evolutionary analyses were conducted in MEGA11.

For the amino acid sequence phylogenetic tree, the nucleotide sequences were translated using MEGA 11, assembled and aligned using the multiple sequence alignment program ClustalX version 2.0.12 and alignment errors were corrected using BioEdit 7.2. The evolutionary history was inferred by using the Maximum Likelihood method and Le_Gascuel model [25]. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 1.5010)). The rate variation model allowed for some sites to be evolutionarily invariable. There was a total of 8750 positions in the final dataset. Evolutionary analyses were conducted in MEGA 11 software [26,27].

2.3. %GC Content

The %GC content of all the sequences were calculated using Oligo calculator—an online tool of University of California Berkeley accessed at the URL http://mcb.berkeley.edu/labs/krantz/tools/oligocalc.html accessed on 18 November 2020.

3. Results

3.1. Overall Patterns of Horizontal Gene Transfer (HGT)

We analysed 221 tnaA gene sequences of all organisms retrieved from the NCBI database. As the presence of tnaA confers unique abilities to the host (particularly bacteria), we addressed the enduring question regarding the distribution and availability of tnaA across known taxonomic groups. Eighty-five prokaryote species have been reported to possess tnaA and/or its activity [5], and indole was considered to be synthesized exclusively by bacteria. Using a phylogenetic approach, we assessed the evolutionary patterns of tnaA. A total of 190 species, that is, 117 bacteria, 36 archaea, 16 fungi, and 21 eukaryotic species harboured the tnaA gene (Table 1). The nucleotide based trees were constructed to elucidate the diversity of the tnaA gene in the selected groups wherein the tnaA gene is susceptible to point mutations and can differentiate gene diversity in closely related organisms. The phylogenetic tree results were further corroborated using %GC content as a parametric method. Due to high variability in GC content of the prokaryotic genomes, internal factors such as GC parametric comparison can help infer horizontal gene transfers [28,29].

In a phylogenetic reconstruction using a dataset containing the 221 tnaA nucleotides and their respective amino acid orthologs, γ-proteobacteria tnaA formed a statistically well-supported branch in both nucleotide and amino acid trees of Enterobacteriales and Vibrionales. Thus, it appears the order ‘Enterobacterales’, γ–proteobacteria have remained conserved in terms of tnaA gene sharing. We noticed several cases of the “non-vertical” appearance of tnaA in distantly related organisms including fungi and other unicellular eukaryotes (Figure 1). Phylogenetic analysis revealed that HGT of tnaA in eukaryotes was not the result of contamination. A wider distribution of tnaA was found in prokaryotes, particularly γ-proteobacteria. The diversity of tnaA followed phylogenetic relationships already established using unique features of rRNA sequences, according to which archaea, bacteria, and eukaryotes are segregated in isolated clades. However, tnaA based phylogeny revealed some random appearances of closely related gene sequences belonging to taxonomically diverse groups revealing HGTs (Figure 1).

The close sequence similarity of >73% for 99% sequence coverage and similar %GC content (Table S1) between Yokenella and Rodentibacter tnaA indicates a possibility of HGT between two distantly related organisms (Figure 1 and Figure 2), which belong to recently diverged sister orders Enterobacteriales and Pasteurellales of the class γ–proteobacteria, respectively. Previously, glg genes were found to undergo complex gene transfers among γ–proteobacteria and other main bacterial groups [30]. Yokenella regensburgei possess a tnaA gene sequence, which contrasts with reports that the organism is negative for indole production [31,32]. This apparent contradiction may be due to the presence of non-functional tnaA, and therefore needs more investigation. This is also observed in Shigella. sp strains which possess the tnaA gene but mutations in cluster 1 and cluster 3 of the tnaA operon consequently render them indole-negative [33]. On the other hand, Enterococcus faecalis and Lactobacillus reuteri produce indole (in vitro), which is indicative of tnaA activity (Table 1); however, the presence of tnaA within their genomes has yet to be determined. Recently, Lactobacillus sp. isolated from infant faeces were studied for their anti-obesity potential [34]. There is an inverse correlation between indole production and fat content which partially confirms functional presence of tryptophanase in Lactobacillus sp. [13].

3.2. TnaA Gene in Archaea

In archaea, the topology of tnaA phylogeny suggests a distinct but common bacterial ancestor (Figure 1 and Figure 2). Few thermophiles thrive within biofilms by secreting extracellular polymeric substances [35], and few halophiles that produce acyl-homoserine lactone (a quorum-sensing signal molecule) secrete extracellular polymeric substances and form biofilms (Table 2). Relationships among genera remained largely undisturbed. Sequences belonging to archaea—‘Halobacteriales, Haloferacales and Natrialbales’ belonging to the class Haloarchaea and other species formed a distinct clade, while the position of the thermophile ‘Aeropyrum’ shifted slightly (Figure 1) with varying %GC content from the archaeal cluster (Table 3, (a)). The exact benefit of indole production by archaea has yet to be determined. Although archaeal biofilms are poorly studied and characterized, increasing evidence suggests that like prokaryotes, archaea benefit from living in biofilms because they afford protection against environmental stresses, fluctuating pH, and toxic chemicals [36,37,38]. Furthermore, polymicrobial biofilms provide environments that facilitate the transfer of genetic material and enable syntrophic associations [35]. The appearance of Aeropyrum pernix in different clades reveals a distinct yet unknown origin, as it is the only representative thermophile that possesses tnaA. The proximity of the Natrialbales clade with those of Halobacteriales and Haloferacales indicates a common ancestry (Figure 1 and Figure 2). Indeed, Haloterrigena spp., which was previously categorized as a member of the Halobacteriaceae family, shows a close relationship with Natrinema spp. and was proposed to be included in the family Natrialbaceae [39] and supported by similar %GC content (Table S1). Interestingly, haloarchaea retained tnaA and other archaea lost the gene. Salinibacter ruber was found near the archaeal cluster (Figure 1) and has similar %GC content (Table 3, (b)) which further supports a common ancestry with halophilic characteristics [40]. TnaA is one of the genes extensively exchanged among members of the Halobacteriaceae and Salinibacter families. Salinibacter ruber was found to be indole-negative which is a common characteristic in most Halobacteriaceae species (Table 1) which further bolsters the case for common ancestry. Further study is required to decipher the influence of indole on the halophilic lifestyles of microbes and the survival strategies of the indole-negative biofilm-producing archaeal microbes.

3.3. Significance of tnaA in the Eukaryotic Life Cycle

For HGT in cases where a wider distribution of genes is found in donor and recipient lineages (and in other taxa), phylogenetic trees are used to investigate transfer polarity [41]. When a gene is only found in donor and recipient groups/taxa, it is generally assumed that the source of gene transfer must be the taxon displaying the most diverse representation of that gene, since the possibility of gene transfer in several organisms from a single species at the same time is unlikely [42]. HGT is an essential evolutionary tool and was thought to be limited to prokaryotes, but over the past decade, increasing evidence indicates genetic materials are exchanged between prokaryotes and eukaryotes (both endosymbionts and free-living organisms) [43,44,45]. The absence of tnaA in the genomes of vertebrates, including mammals, indicates the need for tryptophan degradation has reduced and that the function is largely provided by gut microbes as a result of co-evolution in a holobiont [46]. However, vertebrates have also adopted other means of tryptophan degradation via the kynurenine pathway by cleaving the indole moiety with tryptophan 2,3-dioxygenase in the liver [47].

It has been reported that about 145 genes in man originated from bacteria, including a few involved in amino-acid metabolism acquired by HGT [46]. For instance, Maximum Likelihood and Bayesian phylogenetic methods showed that eukaryotic pyruvate formate lyase, a homolog of the firmicutes gene, may have been acquired through HGT [48]. Similarly, firmicutes (Anaerotruncus, Clostridium, Paraclostridium and Enterocloster) were found within the cluster accommodating fungi and/or unicellular eukaryotes in the nucleotide sequence tree (Figure 1 and Figure 2). TnaA appears to have transferred from bacteria into fungi by two independent HGT events as evidenced by the formation of a distinct clade by members of Sordariomycetes and Eurotiomycetes (Figure 1 and Figure 2) corroborated by their similar %GC content (Table 3, (c)). Sordariomycetes (a clade of fungi—Colletotrichum, Fusarium, Podospora, and Metarhizium) appeared as a sister group of a Blastocystis branch (with high statistical support—based on bootstrap values) (Figure 1 and Figure 2) and similar %GC content (Table 3, (d)). Other related fungi belonging to the Ascomycota group (Aspergillus, Penicillium, and Trichoderma) also had a similar origin as their tnaA sequences were also found to be associated with a clade containing a few protozoa Naegleria and Dictyostelium purpureum (Figure 1). The abundance of several bacterial tnaA genes indicates that the origin of tnaA could be interleaved with members of different bacterial phyla, and endosymbiosis of tnaA possessing bacteria might be the reason for their common ancestry [43]. E. histolytica tnaA might be a result of HGT from a relative of the anaerobic bacterium Fusobacterium (Figure 1 and Figure 2) seconded with similar %GC content (Table 3, (e)). In contrast, the T. vaginalis appears to acquire the gene from a separate origin by HGT from a relative of the anaerobic Bacteroides group (Figure 1 and Figure 2) and similar %GC content (Table 3, (f)). Only three species of anaerobic protists, Trichomonas vaginalis, Tritrichomonas foetus and E. histolytica have been reported to produce indole [49,50].

HGT might have played a role in the evolution and symbiotic adaptation of various unicellular organisms, including eukaryotes. Most of the unicellular organisms (>30%) we studied are pathogenic, and in these organisms, tnaA provides unique advantages for living within a host [20,23]. Previously, Pfam-based analysis led to the identification of 18 HGT events from bacteria to Dictyostelium, which possesses two β-eliminating lyases [51]. Thus, it is evident that the transfer of genetic material occurs between prokaryotes and unicellular eukaryotes, and that these eukaryotes gain a competitive advantage. Indeed, genes of prokaryotic origin are commonly transferred multiple times between protists [43].

HGT events can be classified into two broad types; those that maintain pre-existing functions (maintenance transfers) and those that add new functionality, such as host interaction, defence and adaptation to extreme environments, to the recipient (innovative transfers) [52]. Of note, many lineages (e.g., amoebae, ciliates, dinoflagellates, and non-parasitic excavates) lack reports of HGT events, probably because of sampling bias. Fungi, especially Ascomycota, are prolific producers of indole alkaloids, many of which display potent biological activities [53]. However, the function of indole and its metabolites are still largely undetermined in fungi. The NCBI-BLAST sequence similarities between Fusarium (along with Penicillium, Aspergillus, and Trichoderma) and Porphyromonas, Prevotella, and Trichomonas exceed 60% with e-values of <3 × 10⁻²¹ and similar %GC content (Table S1), indicating a possible HGT from a similar prokaryotic ancestor of the order ‘Bacteroidales’.

Pyruvate can be produced during amino acid metabolism and is specifically required for the regeneration of NAD+ during anaerobic fermentation, whereas aerobic fermentation increases ATP generation via the TCA cycle [1,54,55]. The inclusion of tnaA in a genome broadens metabolic ability especially concerning carbon (gluconeogenesis), amino acid, and nitrogen metabolism [1]. The decomposition and grazing activities of protozoans contribute toward the nitrogen cycle via ammonia generation. Pyruvate and ATP may also be produced via other metabolic pathways and thus the addition of tryptophan conversion may merely result in HGT being considered a ‘maintenance transfer’. Bacteria and protists with such alternative pathways harbouring tnaA thereby gain additional biochemical activity that provides survival and competitive advantages. Tryptophan may also serve as an energy source in S. salmonicida due to the presence of three copies of a bacteria-like TnaA that generate pyruvate, indole, and NH₃ from tryptophan [56]. ‘Indole’ plays several other roles in the ecosystem and is considered an ‘archetypical hormone’ as it can regulate the behaviours of prokaryotes [8] and eukaryotes, including higher vertebrates and plants [23]. Therefore, the production of indole by tnaA may provide a means of manipulating a neighbour’s behaviour, which is of particular use in the gut where bacteria dominate, and other taxonomically diverse groups must compete for nutrients. Thus, the ability to produce indole provides distinct advantages, which suggests horizontal tnaA transfer should be considered an ‘innovative transfer’. The transfer of genetic material in this way is considered an indispensable driver of the evolution of fungi dwelling in the gut of higher organisms [57]. In addition, fungi belonging to the Ascomycota phylum produce indole alkaloids, which have high bioactivities, and octopuses and other marine organisms can use indole to synthesize pigments (e.g., Tyrian purple). The muricid mollusc Dicathais orbita produces Tyrian purple with the help of tissue dwelling Vibrio sp. [18]

Tryptophan is an essential amino acid produced by the shikimic acid pathway (by bacteria and plants, but not animals). Indole is an intermediate of this pathway and may serve as a precursor for tryptophan synthesis [58]. Furthermore, the ability of tnaA to work reversibly gives credence to the notion that bacteria can modulate its ability to produce tryptophan or degrade it to pyruvate and indole. Interestingly, fungi and endophytes interact symbiotically with host plants by synthesizing indole acetic acid (a plant growth hormone).

Table 1. Organisms used in the study and their classification and indole production + positive; − negative; n/a—not available; v—varying.

Organism	Classification (Class; Order; and Family)	Indole Production	Reference	Organism	Classification (Class; Order; and Family)	Indole Production	Reference
				Gram−negative bacteria
A. caviae	Gammaproteobacteria; Aeromonadales; Aeromonadaceae	+	[59]	M. viscosa	Gammaproteobacteria; Alteromonadales; Moritellaceae	−	[60]
A. dhakensis		+	[61]	O. splanchnicus	Bacteroidia; Bacteroidales; Odoribacteraceae	n/a
A. hydrophila		+	[59]	P. ananatis	Gammaproteobacteria; Enterobacterales; Erwiniaceae	+	[62]
A. media		+	[59]	P. stewartia		+	[63]
A. salmonicida		+	[64]	P. laumondii	Gammaproteobacteria; Enterobacterales; Morganellaceae	−	[65]
A. veronii		+	[59]	P. luminescens		−	[65]
A. actinomycetemcomitans	Gammaproteobacteria; Pasteurellales; Pasteurellaceae	n/a		P. shigelloides	Gammaproteobacteria; Enterobacterales; Enterobacterales incertae sedis	+	[66]
A. muciniphila	Verrucomicrobiae; Verrucomicrobiales; Akkermansiaceae	n/a		P. gingivalis	Bacteroidia; Bacteroidales; Porphyromonadaceae	+	[67]
A. wodanis	Gammaproteobacteria; Vibrionales; Vibrionaceae	+	[68]	P. gulae		+	[69]
B. cellulosilyticus	Bacteroidia; Bacteroidales; Bacteroidaceae	n/a		P. intermedia	Bacteroidia; Bacteroidales; Prevotellaceae	+	[70]
B. eggerthii		+	[71]	P. vulgaris	Gammaproteobacteria; Enterobacterales; Morganellaceae	+	[72]
B. faecis		+	[73]	P. alcalifaciens	Gammaproteobacteria; Enterobacterales; Morganellaceae	+	[74]
B. intestinalis		+	[75]	P. rettgeri		+	[76]
B. ovatus		+	[73]	P. stuartii		+	[76]
B. salyersiae		+	[73]	R. ornithinolytica	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	+	[77]
B. stercoris		+	[78]	R. capsulatus	Alphaproteobacteria; Rhodobacterales; Rhodobacteraceae	n/a
B. thetaiotaomicron		+	[73]	R. palustris	Alphaproteobacteria; Rhizobiales; Bradyrhizobiaceae	n/a
B. uniformis		+	[71]	R. pneumotropicus	Gammaproteobacteria; Pasteurellales; Pasteurellaceae	+	[79]
B. hyodysenteriae	Spirochaetia; Brachyspirales; Brachyspiraceae	+	[80]	S. ruber	Bacteroidetes Order II. Incertae sedis; Rhodothermaceae	−	[81]
C. violaceum	Betaproteobacteria; Neisseriales; Chromobacteriaceae	+	[82]	S. boydii	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	−	[83]
C. indologenes	Flavobacteriia; Flavobacteriales; Weeksellaceae	−	[84]	S. dysenteriae		−	[33]
C. amalonaticus	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	n/a		S. flexneri		−	[33]
C. koseri		+	[85]	S. sonnei		−	[33]
C. portucalensis		−	[86]	V. alginolyticus	Gammaproteobacteria; Vibrionales; Vibrionaceae	+	[68]
C.dublinensis	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	+	[87]	V. anguillarum		v	[68]
D. dadantii	Gammaproteobacteria; Enterobacterales; Pectobacteriaceae	+	[88]	V. campbellii		+	[68]
D. dianthicola		+	[89]	V. cholerae		+	[90]
D. solani		+	[91]	V. coralliilyticus		+	[92]
D. zeae		+	[89]	V. crassostreae		+	[93]
E. piscicida	Gammaproteobacteria; Enterobacterales; Hafniaceae	+	[94]	V. cyclitrophicus		−	[68]
E. tarda		+	[94]	V. diabolicus		+	[95]
E. anophelis	Flavobacteriia; Flavobacteriales; Weeksellaceae	+	[96]	V. fluvialis		+	[68]
E. meningoseptica		+	[97]	V. furnissii		v	[98]
E. miricola		+	[97]	V. jasicida		+	[99]
E. norvegicus	Gammaproteobacteria; Vibrionales; Vibrionaceae	+	[100]	V. kanaloae		+	[93]
E. albertii	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	+	[101]	V. lentus		+	[68]
E. coli		+	[102]	V. metoecus		+	[103]
E. fergusonii		+	[104]	V. mimicus		+	[68]
E. marmotae		−	[105]	V. nigripulchritudo		+	[68]
F. hwasookii	Fusobacteria; Fusobacteriales; Fusobacteriaceae	+	[106]	V. owensii		+	[99]
F. necrophorum		+	[107]	V. rotiferianus		+	[99]
F. nucleatum		+	[106]	V. splendidus		+	[68]
F. nucleatum		+	[106]	V. tasmaniensis		+	[68]
G. hollisae	Gammaproteobacteria; Vibrionales; Vibrionaceae	+	[108]	V. vulnificus		+	[68]
H. haemolyticus	Gammaproteobacteria; Pasteurellales; Pasteurellaceae	+	[109]	X. bovienii	Gammaproteobacteria; Enterobacterales; Morganellaceae	n/a
H. influenzae		+	[109]	Y. enterocolitica	Gammaproteobacteria; Enterobacterales; Yersiniaceae	+	[110]
H. parainfluenzae		+	[111]	Y. frederiksenii		+	[112]
H. somni	Gammaproteobacteria; Pasteurellales; Pasteurellaceae	+	[113]	Y. intermedia		+	[114]
K. michiganensis	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	+	[115]	Y. kristensenii		v	[116]
K. oxytoca		+	[117]	Y. massiliensis		+	[118]
Ladecarboxylata	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	+	[119]	Y. regensburgei	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	−	[31]
				Gram−positive bacteria
A. colihominis	Clostridia; Clostridiales; Ruminococcaceae	+	[120]	P. bifermentans	Clostridia; Clostridiales; Peptostreptococcaceae	+	[121]
C. novyi	Clostridia; Clostridiales; Clostridiaceae	+	[122]	S. erythraea	Actinobacteria; Pseudonocardiales; Pseudonocardiaceae	n/a
C. tetani		+	[123]	S. scabiei	Actinobacteria; Streptomycetales; Streptomycetaceae	n/a
C. acnes	Actinobacteria; Propionibacteriales; Propionibacteriaceae	−	[124]	T. denticola	Spirochaetia; Spirochaetales; Spirochaetaceae	+	[125]
E. clostridioformis	Clostridia; Clostridiales; Lachnospiraceae	n/a		T. phagedenis		+	[126]
P. sordellii	Clostridia; Clostridiales; Peptostreptococcaceae	n/a
				Archaea
A. pernix	Thermoprotei; Desulfurococcales; Desulfurococcaceae	n/a		H. larsenii	Halobacteria; Halobacteriales; Halobacteriaceae	n/a
H. jeotgali	Halobacteria; Halobacteriales; Halobacteriaceae	v	[127]	H. pelagica	Halobacteria; Halobacteriales; Halobacteriaceae	+	[128]
H. sulfurireducens	Halobacteria; Halobacteriales; Halobacteriaceae	n/a		H. daqingensis	Halobacteria; Natrialbales; Natrialbaceae	−	[129]
H. hispanica	Halobacteria; Halobacteriales; Haloarculaceae	v	[130]	H. jeotgali	Halobacteria; Natrialbales; Natrialbaceae Halobacteria; Natrialbales; Natrialbaceae	+	[131]
H. marismortui		−	[132]	H. turkmenica		+	[133]
H. taiwanensis		−	[134]	Natrarchaeobaculum sulfurireducens		−	[135]
Haloarcula sp.		−	[134]	N. magadii	Halobacteria; Natrialbales; Natrialbaceae	n/a
H. hubeiense	Halobacteria; Halobacteriales; Halobacteriaceae	n/a		N. pallidum	Halobacteria; Natrialbales; Natrialbaceae	−	[136]
H. salinarum		v	[137]	N. pellirubrum	Halobacteria; Natrialbales; Natrialbaceae Halobacteria; Natrialbales; Natrialbaceae	−	[136]
Halobacterium sp.		n/a		N. versiforme		+	[138]
H. lacisalsi	Halobacteria; Natrialbales; Natrialbaceae	−	[139]	Natrinema sp.		v
H. alexandrinus	Halobacteria; Haloferacales; Haloferacaceae	+	[140]	N. gregoryi		n/a
H. gibbonsii		+	[130]	N. occultus	Halobacteria; Natrialbales; Natrialbaceae	n/a
H. mediterranei		+	[141]	N. aibiense	Halobacteria; Natrialbales; Natrialbaceae	+	[142]
H. volcanii		+	[143]	N. bangense		+	[144]
H. borinquense	Halobacteria; Haloferacales; Haloferacaceae	+	[145]
H. xanaduensis	Halobacteria; Halobacteriales; Halobacteriaceae	−	[146]
				Eukaryotes
		Protists
A. aculeatinus	Eurotiomycetes; Eurotiales; Aspergillaceae	n/a		N. gruberi	Heterolobosea; Schizopyrenida; Vahlkampfiidae	n/a
A. aculeatus		n/a		T. vaginalis	--; Trichomonadida; Trichomonadidae	+	[49]
A. brunneoviolaceus		n/a		E. dispar	Amoebozoa; Mastigamoebida; Entamoebidae Amoebozoa; Mastigamoebida; Entamoebidae	n/a
A. japonicus		n/a		E. histolytica		+	[50]
A. saccharolyticus		n/a		E. invadens		n/a
A. uvarum		n/a		E. nuttalli		n/a
C. graminicola	Sordariomycetes; Glomerellales; Glomerellaceae	n/a
F. fujikuroi	Sordariomycetes; Hypocreales; Nectriaceae	n/a		Higher eukaryotes
F. proliferatum		n/a		O. bimaculoides	Cephalopoda; Octopoda; Octopodidae Cephalopoda; Octopoda; Octopodidae Anthozoa; Scleractinia; Acroporidae	n/a
F. vanettenii		n/a		O. vulgaris		n/a
M. acridum	Sordariomycetes; Hypocreales; Clavicipitaceae	n/a		A. digitifera	Anthozoa; Scleractinia; Acroporidae Anthozoa; Scleractinia; Pocilloporidae Anthozoa; Scleractinia; Acroporidae Anthozoa; Scleractinia; Pocilloporidae	n/a
M. brunneum		n/a		A. millepora		n/a
M. robertsii		n/a		S. pistillata	Anthozoa; Actiniaria; Aiptasiidae	n/a
P. chrysogenum	Eurotiomycetes; Eurotiales; Aspergillaceae	n/a		E. pallida	Anthozoa; Actiniaria; Aiptasiidae	n/a
P. anserina	Sordariomycetes; Sordariales; Chaetomiaceae	n/a		A. tenebrosa	Anthozoa; Actiniaria; Actiniidae	n/a
T. virens	Sordariomycetes; Hypocreales; Hypocreaceae	n/a		N. vectensis	Anthozoa; Actiniaria; Edwardsiidae	n/a
		O. faveolata	Anthozoa; Scleractinia; Merulinidae		n/a
M. neglectum	Chlorophyceae; Sphaeropleales; Selenastraceae	n/a		P. damicornis	Anthozoa; Scleractinia; Pocilloporidae	n/a
D. purpureum	Eumycetozoa; Dictyosteliales; Dictyosteliaceae	n/a		S. kowalevskii	Enteropneusta; --; Harrimaniidae [Hemichordata]	n/a
B. hominis,	Bigyra; Opalinata; Blastocystidae	n/a		L. polyphemus	Merostomata; Xiphosura; Limulidae	n/a
Blastocystis sp.		n/a		F. candida	Collembola; Entomobryomorpha; Isotomidae	n/a
P. tricornutum	Bacillariophyceae; Naviculales; Phaeodactylaceae	n/a		D. melanogaster	Insecta; Diptera; Drosophilidae	n/a
				P. caudatus	Priapulimorpha; Priapulimorphida; Priapulidae	n/a

Table 1. Organisms used in the study and their classification and indole production + positive; − negative; n/a—not available; v—varying.

Organism	Classification (Class; Order; and Family)	Indole Production	Reference	Organism	Classification (Class; Order; and Family)	Indole Production	Reference
				Gram−negative bacteria
A. caviae	Gammaproteobacteria; Aeromonadales; Aeromonadaceae	+	[59]	M. viscosa	Gammaproteobacteria; Alteromonadales; Moritellaceae	−	[60]
A. dhakensis		+	[61]	O. splanchnicus	Bacteroidia; Bacteroidales; Odoribacteraceae	n/a
A. hydrophila		+	[59]	P. ananatis	Gammaproteobacteria; Enterobacterales; Erwiniaceae	+	[62]
A. media		+	[59]	P. stewartia		+	[63]
A. salmonicida		+	[64]	P. laumondii	Gammaproteobacteria; Enterobacterales; Morganellaceae	−	[65]
A. veronii		+	[59]	P. luminescens		−	[65]
A. actinomycetemcomitans	Gammaproteobacteria; Pasteurellales; Pasteurellaceae	n/a		P. shigelloides	Gammaproteobacteria; Enterobacterales; Enterobacterales incertae sedis	+	[66]
A. muciniphila	Verrucomicrobiae; Verrucomicrobiales; Akkermansiaceae	n/a		P. gingivalis	Bacteroidia; Bacteroidales; Porphyromonadaceae	+	[67]
A. wodanis	Gammaproteobacteria; Vibrionales; Vibrionaceae	+	[68]	P. gulae		+	[69]
B. cellulosilyticus	Bacteroidia; Bacteroidales; Bacteroidaceae	n/a		P. intermedia	Bacteroidia; Bacteroidales; Prevotellaceae	+	[70]
B. eggerthii		+	[71]	P. vulgaris	Gammaproteobacteria; Enterobacterales; Morganellaceae	+	[72]
B. faecis		+	[73]	P. alcalifaciens	Gammaproteobacteria; Enterobacterales; Morganellaceae	+	[74]
B. intestinalis		+	[75]	P. rettgeri		+	[76]
B. ovatus		+	[73]	P. stuartii		+	[76]
B. salyersiae		+	[73]	R. ornithinolytica	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	+	[77]
B. stercoris		+	[78]	R. capsulatus	Alphaproteobacteria; Rhodobacterales; Rhodobacteraceae	n/a
B. thetaiotaomicron		+	[73]	R. palustris	Alphaproteobacteria; Rhizobiales; Bradyrhizobiaceae	n/a
B. uniformis		+	[71]	R. pneumotropicus	Gammaproteobacteria; Pasteurellales; Pasteurellaceae	+	[79]
B. hyodysenteriae	Spirochaetia; Brachyspirales; Brachyspiraceae	+	[80]	S. ruber	Bacteroidetes Order II. Incertae sedis; Rhodothermaceae	−	[81]
C. violaceum	Betaproteobacteria; Neisseriales; Chromobacteriaceae	+	[82]	S. boydii	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	−	[83]
C. indologenes	Flavobacteriia; Flavobacteriales; Weeksellaceae	−	[84]	S. dysenteriae		−	[33]
C. amalonaticus	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	n/a		S. flexneri		−	[33]
C. koseri		+	[85]	S. sonnei		−	[33]
C. portucalensis		−	[86]	V. alginolyticus	Gammaproteobacteria; Vibrionales; Vibrionaceae	+	[68]
C.dublinensis	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	+	[87]	V. anguillarum		v	[68]
D. dadantii	Gammaproteobacteria; Enterobacterales; Pectobacteriaceae	+	[88]	V. campbellii		+	[68]
D. dianthicola		+	[89]	V. cholerae		+	[90]
D. solani		+	[91]	V. coralliilyticus		+	[92]
D. zeae		+	[89]	V. crassostreae		+	[93]
E. piscicida	Gammaproteobacteria; Enterobacterales; Hafniaceae	+	[94]	V. cyclitrophicus		−	[68]
E. tarda		+	[94]	V. diabolicus		+	[95]
E. anophelis	Flavobacteriia; Flavobacteriales; Weeksellaceae	+	[96]	V. fluvialis		+	[68]
E. meningoseptica		+	[97]	V. furnissii		v	[98]
E. miricola		+	[97]	V. jasicida		+	[99]
E. norvegicus	Gammaproteobacteria; Vibrionales; Vibrionaceae	+	[100]	V. kanaloae		+	[93]
E. albertii	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	+	[101]	V. lentus		+	[68]
E. coli		+	[102]	V. metoecus		+	[103]
E. fergusonii		+	[104]	V. mimicus		+	[68]
E. marmotae		−	[105]	V. nigripulchritudo		+	[68]
F. hwasookii	Fusobacteria; Fusobacteriales; Fusobacteriaceae	+	[106]	V. owensii		+	[99]
F. necrophorum		+	[107]	V. rotiferianus		+	[99]
F. nucleatum		+	[106]	V. splendidus		+	[68]
F. nucleatum		+	[106]	V. tasmaniensis		+	[68]
G. hollisae	Gammaproteobacteria; Vibrionales; Vibrionaceae	+	[108]	V. vulnificus		+	[68]
H. haemolyticus	Gammaproteobacteria; Pasteurellales; Pasteurellaceae	+	[109]	X. bovienii	Gammaproteobacteria; Enterobacterales; Morganellaceae	n/a
H. influenzae		+	[109]	Y. enterocolitica	Gammaproteobacteria; Enterobacterales; Yersiniaceae	+	[110]
H. parainfluenzae		+	[111]	Y. frederiksenii		+	[112]
H. somni	Gammaproteobacteria; Pasteurellales; Pasteurellaceae	+	[113]	Y. intermedia		+	[114]
K. michiganensis	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	+	[115]	Y. kristensenii		v	[116]
K. oxytoca		+	[117]	Y. massiliensis		+	[118]
Ladecarboxylata	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	+	[119]	Y. regensburgei	Gammaproteobacteria; Enterobacterales; Enterobacteriaceae	−	[31]
				Gram−positive bacteria
A. colihominis	Clostridia; Clostridiales; Ruminococcaceae	+	[120]	P. bifermentans	Clostridia; Clostridiales; Peptostreptococcaceae	+	[121]
C. novyi	Clostridia; Clostridiales; Clostridiaceae	+	[122]	S. erythraea	Actinobacteria; Pseudonocardiales; Pseudonocardiaceae	n/a
C. tetani		+	[123]	S. scabiei	Actinobacteria; Streptomycetales; Streptomycetaceae	n/a
C. acnes	Actinobacteria; Propionibacteriales; Propionibacteriaceae	−	[124]	T. denticola	Spirochaetia; Spirochaetales; Spirochaetaceae	+	[125]
E. clostridioformis	Clostridia; Clostridiales; Lachnospiraceae	n/a		T. phagedenis		+	[126]
P. sordellii	Clostridia; Clostridiales; Peptostreptococcaceae	n/a
				Archaea
A. pernix	Thermoprotei; Desulfurococcales; Desulfurococcaceae	n/a		H. larsenii	Halobacteria; Halobacteriales; Halobacteriaceae	n/a
H. jeotgali	Halobacteria; Halobacteriales; Halobacteriaceae	v	[127]	H. pelagica	Halobacteria; Halobacteriales; Halobacteriaceae	+	[128]
H. sulfurireducens	Halobacteria; Halobacteriales; Halobacteriaceae	n/a		H. daqingensis	Halobacteria; Natrialbales; Natrialbaceae	−	[129]
H. hispanica	Halobacteria; Halobacteriales; Haloarculaceae	v	[130]	H. jeotgali	Halobacteria; Natrialbales; Natrialbaceae Halobacteria; Natrialbales; Natrialbaceae	+	[131]
H. marismortui		−	[132]	H. turkmenica		+	[133]
H. taiwanensis		−	[134]	Natrarchaeobaculum sulfurireducens		−	[135]
Haloarcula sp.		−	[134]	N. magadii	Halobacteria; Natrialbales; Natrialbaceae	n/a
H. hubeiense	Halobacteria; Halobacteriales; Halobacteriaceae	n/a		N. pallidum	Halobacteria; Natrialbales; Natrialbaceae	−	[136]
H. salinarum		v	[137]	N. pellirubrum	Halobacteria; Natrialbales; Natrialbaceae Halobacteria; Natrialbales; Natrialbaceae	−	[136]
Halobacterium sp.		n/a		N. versiforme		+	[138]
H. lacisalsi	Halobacteria; Natrialbales; Natrialbaceae	−	[139]	Natrinema sp.		v
H. alexandrinus	Halobacteria; Haloferacales; Haloferacaceae	+	[140]	N. gregoryi		n/a
H. gibbonsii		+	[130]	N. occultus	Halobacteria; Natrialbales; Natrialbaceae	n/a
H. mediterranei		+	[141]	N. aibiense	Halobacteria; Natrialbales; Natrialbaceae	+	[142]
H. volcanii		+	[143]	N. bangense		+	[144]
H. borinquense	Halobacteria; Haloferacales; Haloferacaceae	+	[145]
H. xanaduensis	Halobacteria; Halobacteriales; Halobacteriaceae	−	[146]
				Eukaryotes
		Protists
A. aculeatinus	Eurotiomycetes; Eurotiales; Aspergillaceae	n/a		N. gruberi	Heterolobosea; Schizopyrenida; Vahlkampfiidae	n/a
A. aculeatus		n/a		T. vaginalis	--; Trichomonadida; Trichomonadidae	+	[49]
A. brunneoviolaceus		n/a		E. dispar	Amoebozoa; Mastigamoebida; Entamoebidae Amoebozoa; Mastigamoebida; Entamoebidae	n/a
A. japonicus		n/a		E. histolytica		+	[50]
A. saccharolyticus		n/a		E. invadens		n/a
A. uvarum		n/a		E. nuttalli		n/a
C. graminicola	Sordariomycetes; Glomerellales; Glomerellaceae	n/a
F. fujikuroi	Sordariomycetes; Hypocreales; Nectriaceae	n/a		Higher eukaryotes
F. proliferatum		n/a		O. bimaculoides	Cephalopoda; Octopoda; Octopodidae Cephalopoda; Octopoda; Octopodidae Anthozoa; Scleractinia; Acroporidae	n/a
F. vanettenii		n/a		O. vulgaris		n/a
M. acridum	Sordariomycetes; Hypocreales; Clavicipitaceae	n/a		A. digitifera	Anthozoa; Scleractinia; Acroporidae Anthozoa; Scleractinia; Pocilloporidae Anthozoa; Scleractinia; Acroporidae Anthozoa; Scleractinia; Pocilloporidae	n/a
M. brunneum		n/a		A. millepora		n/a
M. robertsii		n/a		S. pistillata	Anthozoa; Actiniaria; Aiptasiidae	n/a
P. chrysogenum	Eurotiomycetes; Eurotiales; Aspergillaceae	n/a		E. pallida	Anthozoa; Actiniaria; Aiptasiidae	n/a
P. anserina	Sordariomycetes; Sordariales; Chaetomiaceae	n/a		A. tenebrosa	Anthozoa; Actiniaria; Actiniidae	n/a
T. virens	Sordariomycetes; Hypocreales; Hypocreaceae	n/a		N. vectensis	Anthozoa; Actiniaria; Edwardsiidae	n/a
		O. faveolata	Anthozoa; Scleractinia; Merulinidae		n/a
M. neglectum	Chlorophyceae; Sphaeropleales; Selenastraceae	n/a		P. damicornis	Anthozoa; Scleractinia; Pocilloporidae	n/a
D. purpureum	Eumycetozoa; Dictyosteliales; Dictyosteliaceae	n/a		S. kowalevskii	Enteropneusta; --; Harrimaniidae [Hemichordata]	n/a
B. hominis,	Bigyra; Opalinata; Blastocystidae	n/a		L. polyphemus	Merostomata; Xiphosura; Limulidae	n/a
Blastocystis sp.		n/a		F. candida	Collembola; Entomobryomorpha; Isotomidae	n/a
P. tricornutum	Bacillariophyceae; Naviculales; Phaeodactylaceae	n/a		D. melanogaster	Insecta; Diptera; Drosophilidae	n/a
				P. caudatus	Priapulimorpha; Priapulimorphida; Priapulidae	n/a

Table 2. List of archaea possessing the tnaA gene and/or indole producing activity.

Organism	Unique Growth Requirements	Indole		Biofilm (EPS or QS Signal)	References
		Gene (NCBI)	Production
Aeropyrum pernix K1	Thermophile, 90 °C	+	−	−	[147]
Halalkalicoccus jeotgali B3	1.70–5.1 M NaCl	+	+	−	[131]
Halanaeroarchaeum sulfurireducens strain HSR2	3–5 M NaCl	+	NR	−	[148]
Halanaeroarchaeum sulfurireducens strain M27-SA2	3–5 M NaCl	+	NR	−	[149]
Haloarcula hispanica ATCC 33960	1.70–5.1 M NaCl	+	+	EPS	[132] [150]
Haloarcula hispanica N601		+	+/−	EPS
Haloarcula marismortui ATCC 43049		+	−	EPS
Haloarcula taiwanensis strain Taiwanensis		+	NR	NR
Haloarcula sp. CBA1115		+
Halobacterium hubeiense strain JI20-1		+
Halobacterium salinarum NRC-1	3.5–5 M NaCl	+	NR	+
Halobacterium salinarum R1	4.2 M NaCl	+	NR	EPS/AHL
Halobiforma lacisalsi AJ5	>1.7 M NaCl	+	−	−
Haloferax alexandrinus strain wsp1	4.3 M NaCl	+	+	−	[140]
Haloferax gibbonsii strain ARA6	1.7–4.3 M NaCl	+	+	EPS
Haloferax mediterranei ATCC 33500	1–5.2 M NaCl	+	+	EPS
Haloferax volcanii DS2	2.6–4.3 M NaCl	+	NR	+/AHLs
Halogeometricum borinquense DSM 11551	3.4–4.3 M NaCl	+	+	+/AHLs
Halopiger xanaduensis SH-6	4.3 M NaCl	+	−	−
Haloprofundus sp. MHR1	0.9–4.8 M NaCl	+	−	−
Halostagnicola larsenii XH-48	2.5–5.0 M NaCl	+	−	−
Halostella pelagica strain DL-M4	2.6 M NaCl	+	NA	−
Haloterrigena daqingensis strain JX313	1.7–5.5 M NaCl	+	−	−
Haloterrigena jeotgali strain A29	2.6–3.4 M NaCl	+	+	−
Haloterrigena turkmenica DSM 5511	2.6–3.4 M NaCl	+	NA	NA
Natrarchaeobaculum sulfurireducens strain AArc1	3–5 M NaCl	+	−	NA
Natrialba magadii ATCC 43099	3.4 M NaCl	+	NR	NA
Natrinema pallidum strain BOL6-1	3.4–4.3 M NaCl	+	−	NA
Natrinema pellirubrum DSM 15624	3.4–4.3 M NaCl	+	−	NA
Natrinema versiforme strain BOL5-4	3.4–4.3 M NaCl	+	+	NA
Natronobacterium gregoryi SP2	2.0–5 M NaCl	+	NR	NA
Natronococcus occultus SP4	2 M NaCl	+	NR	AHL/Biofilm
Natronorubrum aibiense strain 7-3	2.0–4.3 M NaCl	+	+	NA	[142]
Natronorubrum bangense strain JCM 10635	2.0–4.3 M NaCl	+	+	NA	[144]

NR: not reported; NA: not available; AHL: Acyl-homoserine lactone; EPS: extracellular polymeric substance. + positive; − negative.

Table 2. List of archaea possessing the tnaA gene and/or indole producing activity.

Organism	Unique Growth Requirements	Indole		Biofilm (EPS or QS Signal)	References
		Gene (NCBI)	Production
Aeropyrum pernix K1	Thermophile, 90 °C	+	−	−	[147]
Halalkalicoccus jeotgali B3	1.70–5.1 M NaCl	+	+	−	[131]
Halanaeroarchaeum sulfurireducens strain HSR2	3–5 M NaCl	+	NR	−	[148]
Halanaeroarchaeum sulfurireducens strain M27-SA2	3–5 M NaCl	+	NR	−	[149]
Haloarcula hispanica ATCC 33960	1.70–5.1 M NaCl	+	+	EPS	[132] [150]
Haloarcula hispanica N601		+	+/−	EPS
Haloarcula marismortui ATCC 43049		+	−	EPS
Haloarcula taiwanensis strain Taiwanensis		+	NR	NR
Haloarcula sp. CBA1115		+
Halobacterium hubeiense strain JI20-1		+
Halobacterium salinarum NRC-1	3.5–5 M NaCl	+	NR	+
Halobacterium salinarum R1	4.2 M NaCl	+	NR	EPS/AHL
Halobiforma lacisalsi AJ5	>1.7 M NaCl	+	−	−
Haloferax alexandrinus strain wsp1	4.3 M NaCl	+	+	−	[140]
Haloferax gibbonsii strain ARA6	1.7–4.3 M NaCl	+	+	EPS
Haloferax mediterranei ATCC 33500	1–5.2 M NaCl	+	+	EPS
Haloferax volcanii DS2	2.6–4.3 M NaCl	+	NR	+/AHLs
Halogeometricum borinquense DSM 11551	3.4–4.3 M NaCl	+	+	+/AHLs
Halopiger xanaduensis SH-6	4.3 M NaCl	+	−	−
Haloprofundus sp. MHR1	0.9–4.8 M NaCl	+	−	−
Halostagnicola larsenii XH-48	2.5–5.0 M NaCl	+	−	−
Halostella pelagica strain DL-M4	2.6 M NaCl	+	NA	−
Haloterrigena daqingensis strain JX313	1.7–5.5 M NaCl	+	−	−
Haloterrigena jeotgali strain A29	2.6–3.4 M NaCl	+	+	−
Haloterrigena turkmenica DSM 5511	2.6–3.4 M NaCl	+	NA	NA
Natrarchaeobaculum sulfurireducens strain AArc1	3–5 M NaCl	+	−	NA
Natrialba magadii ATCC 43099	3.4 M NaCl	+	NR	NA
Natrinema pallidum strain BOL6-1	3.4–4.3 M NaCl	+	−	NA
Natrinema pellirubrum DSM 15624	3.4–4.3 M NaCl	+	−	NA
Natrinema versiforme strain BOL5-4	3.4–4.3 M NaCl	+	+	NA
Natronobacterium gregoryi SP2	2.0–5 M NaCl	+	NR	NA
Natronococcus occultus SP4	2 M NaCl	+	NR	AHL/Biofilm
Natronorubrum aibiense strain 7-3	2.0–4.3 M NaCl	+	+	NA	[142]
Natronorubrum bangense strain JCM 10635	2.0–4.3 M NaCl	+	+	NA	[144]

NR: not reported; NA: not available; AHL: Acyl-homoserine lactone; EPS: extracellular polymeric substance. + positive; − negative.

Table 3. The %GC content comparison of (a) Aeropyrum pernix K1 and archaeal cluster, (b) Salinibacter ruber and archaeal cluster, (c) Sadriomycetes and Euratiomycetes cluster, (d) Sadriomycetes and Blastocystis.sp branch, (e) Fusobacterium sp. branch and Entamoeba histolytica HM-1 and (f) Bacteroides.sp branch and Trichomonas vaginalis G3.

(a)		(b)		(c)
Organism	%GC content	Organism	%GC content	Organism	%GC content
N. occultus SP4	68	N. occultus SP4	68	A. aculeatinus CBS 121060	65
N. aibiense strain 7-3	66	N. aibiense strain 7-3	66	A. aculeatus ATCC 16872	63
N. gregoryi SP2	65	N. gregoryi SP2	65	A. brunneoviolaceus CBS 621.78	65
N. pallidum strain BOL6-1	66	N. pallidum strain BOL6-1	66	A. japonicus CBS 114.51	62
N. magadii ATCC 43099	66	N. magadii ATCC 43099	66	A. saccharolyticus JOP 1030-1	65
N. sulfurireducens strain AArc1	67	N. sulfurireducens strain AArc1	67	A. uvarum CBS 121591	64
H. borinquense DSM 11551	63	H. borinquense DSM 11551	63	P. anserina S mat+	56
H. gibbonsii strain ARA6	68	H. gibbonsii strain ARA6	68	M. acridum CQMa 102	53
H. jeotgali strain A29	68	H. jeotgali strain A29	68	M. brunneum ARSEF 3297	52
H. pelagica strain DL-M4	67	H. pelagica strain DL-M4	67	M. robertsii ARSEF 23	53
H. larsenii XH-48	65	H. larsenii XH-48	65	F. fujikuroi IMI 58289	50
H. xanaduensis SH-6	69	H. xanaduensis SH-6	69	F. proliferatum ET1	50
H. lacisalsi AJ5	69	H. lacisalsi AJ5	69	F. vanettenii	58
Halobacterium sp. DL1	71	Halobacterium sp. DL1	71	C. graminicola M1.001	56
H. salinarum NRC-1	71	H. salinarum NRC-1	71
H. sulfurireducens strain HSR2	67	H. sulfurireducens strain HSR2	67
H. jeotgali B3	65	H.jeotgali B3	65
A. pernix K1	56	S. ruber DSM 13855	67
(d)		(e)		(f)
Organism	%GC content	Organism	%GC content	Organism	%GC content
P. anserina S mat+	56	F. hwasookii ChDC	34	B. cellulosilyticus strain WH2	46
M. acridum CQMa 102	53	F. hwasookii ChDC F300	31	B. eggerthii strain NCTC11155	45
M. brunneum ARSEF 3297	52	F. necrophorum strain 1_1_36S	40	B. faecis MAJ27	46
M. robertsii ARSEF 23	53	F. nucleatum ATCC 25586	33	B. intestinalis DSM 17393	46
F. fujikuroi IMI 58289	50	F. nucleatum strain NCTC10562	33	B. ovatus strainBSD2780061688st1_C6	47
F. proliferatum ET1	50	E. histolytica HM-1	33	B. salyersiae CL02T12C01	46
F. vanettenii	58			B. stercoris ATCC 43183	44
C. graminicola M1.001	56			B. thetaiotaomicron strain 7330	48
B. hominis, Singapore isolate B	56			T. vaginalis G3	48
Blastocystis sp. strain WR1	57

Table 3. The %GC content comparison of (a) Aeropyrum pernix K1 and archaeal cluster, (b) Salinibacter ruber and archaeal cluster, (c) Sadriomycetes and Euratiomycetes cluster, (d) Sadriomycetes and Blastocystis.sp branch, (e) Fusobacterium sp. branch and Entamoeba histolytica HM-1 and (f) Bacteroides.sp branch and Trichomonas vaginalis G3.

(a)		(b)		(c)
Organism	%GC content	Organism	%GC content	Organism	%GC content
N. occultus SP4	68	N. occultus SP4	68	A. aculeatinus CBS 121060	65
N. aibiense strain 7-3	66	N. aibiense strain 7-3	66	A. aculeatus ATCC 16872	63
N. gregoryi SP2	65	N. gregoryi SP2	65	A. brunneoviolaceus CBS 621.78	65
N. pallidum strain BOL6-1	66	N. pallidum strain BOL6-1	66	A. japonicus CBS 114.51	62
N. magadii ATCC 43099	66	N. magadii ATCC 43099	66	A. saccharolyticus JOP 1030-1	65
N. sulfurireducens strain AArc1	67	N. sulfurireducens strain AArc1	67	A. uvarum CBS 121591	64
H. borinquense DSM 11551	63	H. borinquense DSM 11551	63	P. anserina S mat+	56
H. gibbonsii strain ARA6	68	H. gibbonsii strain ARA6	68	M. acridum CQMa 102	53
H. jeotgali strain A29	68	H. jeotgali strain A29	68	M. brunneum ARSEF 3297	52
H. pelagica strain DL-M4	67	H. pelagica strain DL-M4	67	M. robertsii ARSEF 23	53
H. larsenii XH-48	65	H. larsenii XH-48	65	F. fujikuroi IMI 58289	50
H. xanaduensis SH-6	69	H. xanaduensis SH-6	69	F. proliferatum ET1	50
H. lacisalsi AJ5	69	H. lacisalsi AJ5	69	F. vanettenii	58
Halobacterium sp. DL1	71	Halobacterium sp. DL1	71	C. graminicola M1.001	56
H. salinarum NRC-1	71	H. salinarum NRC-1	71
H. sulfurireducens strain HSR2	67	H. sulfurireducens strain HSR2	67
H. jeotgali B3	65	H.jeotgali B3	65
A. pernix K1	56	S. ruber DSM 13855	67
(d)		(e)		(f)
Organism	%GC content	Organism	%GC content	Organism	%GC content
P. anserina S mat+	56	F. hwasookii ChDC	34	B. cellulosilyticus strain WH2	46
M. acridum CQMa 102	53	F. hwasookii ChDC F300	31	B. eggerthii strain NCTC11155	45
M. brunneum ARSEF 3297	52	F. necrophorum strain 1_1_36S	40	B. faecis MAJ27	46
M. robertsii ARSEF 23	53	F. nucleatum ATCC 25586	33	B. intestinalis DSM 17393	46
F. fujikuroi IMI 58289	50	F. nucleatum strain NCTC10562	33	B. ovatus strainBSD2780061688st1_C6	47
F. proliferatum ET1	50	E. histolytica HM-1	33	B. salyersiae CL02T12C01	46
F. vanettenii	58			B. stercoris ATCC 43183	44
C. graminicola M1.001	56			B. thetaiotaomicron strain 7330	48
B. hominis, Singapore isolate B	56			T. vaginalis G3	48
Blastocystis sp. strain WR1	57

4. Conclusions

Among prokaryotes, we observed a wider distribution of tnaA in γ-proteobacteria, which share similar copies of the gene among themselves and therefore grouped. Our phylogenetic analysis suggests HGT has played a crucial role in tnaA gene transfer and provided ‘maintenance and innovative gain’ to recipients. These results are quite relevant from the host–parasite interaction perspective. Acquired tnaA has probably enabled unicellular eukaryotes to regulate the gut environment and live in synergy with microbiota and other enteric bacteria. We suggest that the adaptation of the intestinal protozoan Blastocystis sp. to the gut resulted in the lateral acquisition of the tnaA gene and enabled the supply of additional metabolites to the pre-existing metabolic systems.

Similarly, other unicellular eukaryotes such as Dictyostelium, Entamoeba, and Trichomonas may have inherited tnaA from a eukaryotic ancestor. However, the sparse presence of tnaA in higher eukaryotes (e.g., sea anemone, corals, octopuses, drosophila, and nematodes) is insufficient to reveal its origins. On the other hand, the common occurrence of tnaA in archaea belonging to the class Haloarchaea is intriguing and warrants further investigation of the role of indole in adaptation to halophilic environments. Collectively, these results reveal new avenues of research directed toward improved understanding of the roles of indole in various organisms including eukaryotes.