Molecular basis of indole production catalyzed by tryptophanase in the genus Prevotella.

Indole is most commonly known as a diagnostic marker and a malodorous chemorepellent. More recently, it has been recognized that indole also functions as an extracellular signaling molecule that controls bacterial physiology and virulence. The gene (tnaA) for tryptophanase, which produces indole, ammonia, and pyruvate via ß-elimination of L-tryptophan, was cloned from Prevotella intermedia ATCC 25611 and recombinant TnaA was purified and enzymatically characterized. Analysis by reverse transcriptase-mediated PCR showed that the gene was not cotranscribed with flanking genes in P. intermedia. The results of gel-filtration chromatography suggested that P. intermedia TnaA forms homodimers, unlike other reported TnaA proteins. Recombinant TnaA exhibited a K(m) of 0.23 ± 0.01 mM and k(cat) of 0.45 ± 0.01 s(-1). Of 22 Prevotella species tested, detectable levels of indole were present in the culture supernatants of six, including P. intermedia. Southern hybridization showed that tnaA-positive signals were present in the genomic DNA from the six indole-producing strains, but not the other 16 strains tested. The indole-producing strains, with the exception of Prevotella micans, formed a phylogenetic cluster based on trees constructed using 16S rRNA gene sequences, which suggested that tnaA in P. micans might have been transferred from other Prevotella species relatively recently.

Production of indole from L-tryptophan and effects of these compounds on biofilm formation by Fusobacterium nucleatum ATCC 25586.

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.