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Applied and Environmental Microbiology Jul 2010The l-tryptophan degradation product indole is a purported extracellular signaling molecule that influences biofilm formation in various bacteria. Here we analyzed the...
The l-tryptophan degradation product indole is a purported extracellular signaling molecule that influences biofilm formation in various bacteria. Here we analyzed the mechanisms of indole production in Fusobacterium nucleatum and the effects of tryptophan and indole on F. nucleatum planktonic and biofilm cells. The amino acid sequence deduced from the fn1943 gene in F. nucleatum ATCC 25586 was 28% identical to that deduced from tnaA in Escherichia coli, which encodes tryptophanase catalyzing the beta-elimination of l-tryptophan to produce indole. The fn1943 gene was cotranscribed with the downstream gene fn1944, which is a homolog of tnaB encoding low-affinity tryptophan permease. The transcript started at position -68 or -153 from the first nucleotide of the fn1943 translation initiation codon. Real-time quantitative PCR showed that much more F. nucleatum fn1943 transcripts were obtained from log-phase cells than from stationary-phase cells. Indole production by the purified recombinant protein encoded by fn1943 was examined using high-performance liquid chromatography. The K(m) and k(cat) of the enzyme were 0.26 +/- 0.03 mM and 0.74 +/- 0.04 s(-1), respectively. F. nucleatum biofilm formation and the biofilm supernatant concentration of indole increased dose dependently with increasing tryptophan concentrations. Exogenous indole also increased F. nucleatum biofilm formation in a dose-dependent manner. Even at very high concentrations, tryptophan did not affect fn1943 expression, whereas similar indole concentrations decreased expression. Thus, exogenous tryptophan and indole were suggested to increase F. nucleatum biofilms.
Topics: Base Sequence; Biofilms; Fusobacterium nucleatum; Indoles; Molecular Sequence Data; Plankton; Recombinant Proteins; Tryptophan; Tryptophanase
PubMed: 20472741
DOI: 10.1128/AEM.00166-10 -
The Journal of Biological Chemistry May 1991Tryptophanase (tryptophan: indole-lyase) from Escherichia coli has been isolated in the holoenzyme form and its absorption spectra and acid-base chemistry have been...
Tryptophanase (tryptophan: indole-lyase) from Escherichia coli has been isolated in the holoenzyme form and its absorption spectra and acid-base chemistry have been reevaluated. Apoenzyme has been prepared by dialysis against sodium phosphate and L-alanine and molar absorptivities of the coenzyme bands have been estimated by readdition of pyridoxal 5'-phosphate. The spectrophotometric titration curve, whose midpoint is at pH 7.6 in 0.1 M potassium phosphate buffers, indicates some degree of cooperativity in dissociation of a pair of protons. Resolution of the computed spectra of individual ionic forms of the enzyme with lognormal distribution curves shows that band shapes are similar to those of model Schiff bases and of aspartate aminotransferase. Using molar areas from the latter we estimated amounts of individual tautomeric species. In addition to ketoenamine and enolimine or covalent adduct the high pH form also appears to contain approximately 18% of a species with a dipolar ionic ring (protonated on the ring nitrogen and with phenolate -O-). We suggest that this may be the catalytically active form of the coenzyme in tryptophanase. The equilibrium between tryptophanase and L-alanine has also been reevaluated.
Topics: Escherichia coli; Hydrogen-Ion Concentration; Ions; Kinetics; Spectrum Analysis; Tryptophanase
PubMed: 2033039
DOI: No ID Found -
Journal of Microbiology and... Apr 2024Indigo is a valuable, natural blue dye that has been used for centuries in the textile industry. The large-scale commercial production of indigo relies on its extraction...
Indigo is a valuable, natural blue dye that has been used for centuries in the textile industry. The large-scale commercial production of indigo relies on its extraction from plants and chemical synthesis. Studies are being conducted to develop methods for environment-friendly and sustainable production of indigo using genetically engineered microbes. Here, to enhance the yield of bioindigo from an whole-cell system containing tryptophanase (TnaA) and flavin-containing monooxygenase (FMO), we evaluated tryptophan transporters to improve the transport of aromatic compounds, such as indole and tryptophan, which are not easily soluble and passable through cell walls. Among the three transporters, Mtr, AroP, and TnaB, AroP enhanced indigo production the most. The combination of each transporter with AroP was also evaluated, and the combination of AroP and TnaB showed the best performance compared to the single transporters and two transporters. Bioindigo production was then optimized by examining the culture medium, temperature, isopropyl β-D-1-thiogalactopyranoside concentration, shaking speed (rpm), and pH. The novel strain containing and plasmid with and produced 8.77 mM (2.3 g/l) of bioindigo after 66 h of culture. The produced bioindigo was further recovered using a simple method and used as a watercolor dye, showing good mixing with other colors and color retention for a relatively long time. This study presents an effective strategy for enhancing indigo production using a combination of transporters.
Topics: Tryptophan; Escherichia coli; Indoles; Indigo Carmine; Tryptophanase; Escherichia coli Proteins; Culture Media; Oxygenases; Amino Acid Transport Systems; Plasmids; Metabolic Engineering; Fermentation; Hydrogen-Ion Concentration; Coloring Agents; Temperature
PubMed: 38213292
DOI: 10.4014/jmb.2308.08039 -
Journal of Bacteriology Apr 1965Beggs, William H. (University of Cincinnati, Cincinnati, Ohio), and Herman C. Lichstein. Repression of tryptophanase synthesis in Escherichia coli. J. Bacteriol....
Beggs, William H. (University of Cincinnati, Cincinnati, Ohio), and Herman C. Lichstein. Repression of tryptophanase synthesis in Escherichia coli. J. Bacteriol. 89:996-1004. 1965.-The nature of the glucose effect on tryptophanase in Escherichia coli (Crookes) was investigated to test the catabolite-repression hypothesis. Under static conditions of growth in the presence of 0.005 m glucose, tryptophanase was repressed and remained so upon continued static incubation subsequent to glucose exhaustion. Aeration following glucose exhaustion under static cultural conditions resulted in rapid enzyme synthesis. In the absence of glucose, certain amino acids repressed tryptophanase synthesis early in the growth cycle under aerated conditions. An inverse relationship was observed between the concentration of acid-hydrolyzed casein and the level of tryptophanase. At 3 hr, enzyme activity in cells grown in media containing 0.05% acid-hydrolyzed casein was at least five times that of cells grown in the presence of 1% casein. Addition of 0.005 m d- or l-serine to a 0.05% acid-hydrolyzed casein medium rendered the medium capable of strongly repressing tryptophanase. Glucose-expended medium was prepared by allowing cells to grow and exhaust glucose in static culture. When this expended medium was recovered and inoculated with fresh cells not previously exposed to glucose, tryptophanase synthesis was repressed for a short period in shake culture, but in static culture enzyme synthesis was only slightly affected. When the expended medium was prepared from shake cultures, fresh cells were not repressed strongly when subsequent incubation was carried out aerobically. The tryptophan pool in glucose-repressed cells grown in shake culture was appreciably less than in cells grown in the absence of glucose or in cells undergoing synthesis of tryptophanase after exhaustion of the sugar.
Topics: Amino Acids; Carbohydrates; Enzyme Inhibitors; Escherichia coli; Glucose; Lyases; Pharmacology; Research; Serine; Tryptophan; Tryptophanase
PubMed: 14276127
DOI: 10.1128/jb.89.4.996-1004.1965 -
Journal of Bacteriology Jan 1971Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression...
Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression of beta-glactosidase. Induction of tryptophanase and beta-galactosidase in cultures grown with various carbon sources including succinate, glycerol, pyruvate, glucose, gluconate, and arabinose is affected differently by the various carbon sources. The extent of induction does not seem to be related to the growth rate of the culture permitted by the carbon source during the course of the experiment. In cultures grown with glycerol as carbon source, preinduced for beta-galactosidase or tryptophanase and made permeable by ethylenediaminetetraacetic acid (EDTA) treatment, catabolite repression of tryptophanase was not affected markedly by the addition of cAMP (3',5'-cyclic adenosine monophosphate). Catabolite repression by glucose was only partially relieved by the addition of cAMP. In contrast, under the same conditions, cAMP completely relieved catabolite repression of beta-galactosidase by either pyruvate or glucose. Under conditions of limited oxygen, induction of tryptophanase is sensitive to catabolite repression; under the same conditions, beta-galactosidase induction is not sensitive to catabolite repression. Induction of tryptophanase in cells grown with succinate as carbon source is sensitive to catabolite repression by glycerol and pyruvate as well as by glucose. Studies with a glycerol kinaseless mutant indicate that glycerol must be metabolized before it can cause catabolite repression. The EDTA treatment used to make the cells permeable to cAMP was found to affect subsequent growth and induction of either beta-galactosidase or tryptophanase much more adversely in E. coli strain BB than in E. coli strain K-12. Inducation of tryptophanase was reduced by the EDTA treatment significantly more than induction of beta-galactosidase in both strains. Addition of 2.5 x 10(-3)m cAMP appeared partially to reverse the inhibitory effect of the EDTA treatment on enzyme induction but did not restore normal growth.
Topics: Arabinose; Culture Media; Cyclic AMP
PubMed: 4322348
DOI: 10.1128/jb.105.1.303-312.1971 -
Acta Crystallographica. Section F,... Mar 2015Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtained under the same crystallization conditions. Both forms belonged to the...
Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtained under the same crystallization conditions. Both forms belonged to the same space group P43212 but had slightly different unit-cell parameters. The holo crystal form, with pyridoxal phosphate (PLP) bound to Lys270 of both polypeptide chains in the asymmetric unit, diffracted to 2.9 Å resolution. The second crystal form diffracted to 3.2 Å resolution. Of the two subunits in the asymmetric unit, one was found in the holo form, while the other appeared to be in the apo form in a wide-open conformation with two sulfate ions bound in the vicinity of the active site. The conformation of all holo subunits is the same in both crystal forms. The structures suggest that Trpase is flexible in the apo form. Its conformation partially closes upon binding of PLP. The closed conformation might correspond to the enzyme in its active state with both cofactor and substrate bound in a similar way as in tyrosine phenol-lyase.
Topics: Apoenzymes; Catalytic Domain; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Protein Binding; Protein Structure, Secondary; Pyridoxal Phosphate; Tryptophanase
PubMed: 25760702
DOI: 10.1107/S2053230X15000850 -
Applied and Environmental Microbiology Apr 2023The study of fusobacterial virulence factors has dramatically benefited from the creation of various genetic tools for DNA manipulation, including based counterselection...
The study of fusobacterial virulence factors has dramatically benefited from the creation of various genetic tools for DNA manipulation, including based counterselection for in-frame deletion mutagenesis in Fusobacterium nucleatum, which was recently developed. However, this method requires a host lacking the gene, which is an inherent limitation. To circumvent this limitation, we explored the possibility of using the gene that encodes a toxin consisting of a HicAB toxin-antitoxin module in Fusobacterium periodonticum as a new counterselective marker. Interestingly, the full-length gene is not toxic in F. nucleatum, but a truncated gene version lacking the first six amino acids is functional as a toxin. The toxin expression is driven by an promoter and is controlled at its translational level by using a theophylline-responsive riboswitch unit. As a proof of concept, we created markerless in-frame deletions in the fusobacterial adhesin gene within the F. nucleatum operon and the gene that encodes the tryptophanase for indole production. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of colonies grown on theophylline-added plates and resulted in in-frame deletions in 50% of the screened isolates. This -based counterselection system provides a robust and reliable counterselection in wild-type background F. nucleatum and should also be adapted for use in other bacteria. Fusobacterium nucleatum is an indole-producing human oral anaerobe associated with periodontal diseases, preterm birth, and several cancers. Little is known about the mechanisms of fusobacterial pathogenesis and associated factors, mainly due to the lack of robust genetic tools for this organism. Here, we showed that a mutated gene from Fusobacterium periodonticum expresses an active toxin and was used as a counterselection marker. This -based in-frame deletion system efficiently creates in-frame deletion mutations in the wild-type background of F. nucleatum. This is the first report to use the gene as a counterselection marker in a bacterial genetic study.
Topics: Infant, Newborn; Humans; Female; Fusobacterium nucleatum; Theophylline; Premature Birth; Mutation; Toxins, Biological
PubMed: 37039662
DOI: 10.1128/aem.00091-23 -
Frontiers in Microbiology 2021Indole works as an interspecies signal molecule to regulate multiple physiological activities, like antibiotic resistance, acid resistance, and virulence. However, the...
Indole works as an interspecies signal molecule to regulate multiple physiological activities, like antibiotic resistance, acid resistance, and virulence. However, the effect of indole on conjugation is unknown. Here, with λπ as a donor strain that carries a chromosomally integrated conjugative RP4 plasmid, we explored the effect of indole on conjugation of a mobilizable pUCP24T plasmid imparting gentamycin resistance. The results showed that exogenous indole treatment inhibited conjugative transfer of pUCP24T from λπ to recipient strains, and . Furthermore, raising endogenous indole production through overexpression of TnaA, a tryptophanase, in λπ significantly inhibited both λπ- and λπ- conjugation, whereas deficiency of reversed the phenotype. Subsequent mechanistic studies revealed that exogenous indole significantly inhibited the expression of mating pair formation gene () and the DNA transfer and replication gene (), mainly due to the promotion of regulatory genes ( and ), and the result was confirmed in knockout and overexpression strains. Additionally, we found that both extracellular indole production and expression of λπ were downregulated by ciprofloxacin (CIP). Intriguingly, one-eighth minimum inhibitory concentration of CIP treatment clearly facilitated both λπ- and λπ- conjugation, and indole inhibited CIP-induced conjugation frequency. These data suggest that indole may play a negative role in the process of CIP-induced conjugation. This is the first study to reveal the biological function of indole-inhibiting conjugation and its role in CIP-induced conjugation, which may be developed into a new way of controlling the spread of antibiotic resistance.
PubMed: 33815310
DOI: 10.3389/fmicb.2021.628133 -
Journal of Bacteriology Sep 2011Enteropathogenic Escherichia coli(EPEC) requires the tnaA-encoded enzyme tryptophanase and its substrate tryptophan to synthesize diffusible exotoxins that kill the...
Enteropathogenic Escherichia coli(EPEC) requires the tnaA-encoded enzyme tryptophanase and its substrate tryptophan to synthesize diffusible exotoxins that kill the nematode Caenorhabditis elegans. Here, we demonstrate that the RNA-binding protein CsrA and the tryptophan permease TnaB coregulate tryptophanase activity, through mutually exclusive pathways, to stimulate toxin-mediated paralysis and killing of C. elegans.
Topics: Amino Acid Sequence; Amino Acid Transport Systems; Animals; Base Sequence; Caenorhabditis elegans; Enteropathogenic Escherichia coli; Escherichia coli Proteins; Exotoxins; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Operon; RNA-Binding Proteins; Repressor Proteins; Tryptophan; Tryptophanase
PubMed: 21705596
DOI: 10.1128/JB.05197-11 -
Engineering in Life Sciences Aug 2017The hydroxylation of tryptophan is an important reaction in the biosynthesis of natural products. 5-Hydroxytryptophan (5HTP) is not only an important compound for its...
The hydroxylation of tryptophan is an important reaction in the biosynthesis of natural products. 5-Hydroxytryptophan (5HTP) is not only an important compound for its pharmaceutical value but also because it is the precursor of other molecules, such as serotonin. In this study, we have extended the metabolism of an strain to produce 5HTP. Aromatic amino acid hydroxylase from (AAAH) was selected using an structure-based approach. We have predicted and selected several substrate-determining residues using sequence, phylogenetic and functional divergence analyses; we also did rational design on AAAH to shift the enzyme preference from phenylalanine to tryptophan. Whole cell bioconversion assays were used to show the effect of predicted sites. In general, all of them decreased the preference toward phenylalanine and increased the tryptophan synthesis activity. The best performer, AAAH-W192F, was transformed into a strain that had the tryptophanase gene disrupted and carried a human tetrahydrobiopterin (BH4) regeneration pathway. The resulting strain was capable of synthesizing 2.5 mM 5HTP after 24 hours. This work demonstrates the application of computational approaches for protein engineering and further coupling with the bacterial metabolism.
PubMed: 32624837
DOI: 10.1002/elsc.201700064