tnaA Gene Research Articles

Characteristics of the gut microbiota and serum metabolites in postmenopausal women with reduced bone mineral density.

Emerging evidence suggests that the gut microbiota is closely associated with bone homeostasis. However, little is known about the relationships among the bone mineral density (BMD) index, bone turnover markers, and the gut microbiota and its metabolites in postmenopausal women. In this study, to understand gut microbiota signatures and serum metabolite changes in postmenopausal women with reduced BMD, postmenopausal individuals with normal or reduced BMD were recruited and divided into normal and OS groups. Feces and serum samples were collected for 16S rRNA gene sequencing, liquid chromatography coupled with mass spectrometry (LC-MS)-based metabolomics and integrated analysis. The results demonstrated that bacterial richness and diversity were greater in the OS group than in the normal group. Additionally, distinguishing bacteria were found among the two groups and were closely associated with the BMD index and bone turnover markers. Metabolomic analysis revealed that the expression of serum metabolites, such as etiocholanolone, testosterone sulfate, and indole-3-pyruvic acid, and the corresponding signaling pathways, especially those involved in tryptophan metabolism, fatty acid degradation and steroid hormone biosynthesis, also changed significantly. Correlation analysis revealed positive associations between normal group-enriched Bacteroides abundance and normal group-enriched etiocholanolone and testosterone sulfate abundances; in particular, Bacteroides correlated positively with BMD. Importantly, the tryptophan-indole metabolism pathway was uniquely metabolized by the gut bacteria-derived tnaA gene, the predicted abundance of which was significantly greater in the normal group than in the control group, and the abundance of Bacteroides was strongly correlated with the tnaA gene. Our results indicated a clear difference in the gut microbiota and serum metabolites of postmenopausal women. Specifically altered bacteria and derived metabolites were closely associated with the BMD index and bone turnover markers, indicating the potential of the gut microbiota and serum metabolites as modifiable factors and therapeutic targets for preventing osteoporosis.

Biosynthesis of melatonin from l-tryptophan by an engineered microbial cell factory

BackgroundThe demand for melatonin is increasing due to its health-promoting bioactivities such as antioxidant and sleep benefits. Although melatonin is present in various organisms, its low content and high extraction cost make it unsustainable. Biosynthesis is a promising alternative method for melatonin production. However, the ectopic production of melatonin in microorganisms is very difficult due to the low or insoluble expression of melatonin synthesis genes. Hence, we aim to explore the biosynthesis of melatonin using Escherichia coli as a cell factory and ways to simultaneously coordinated express genes from different melatonin synthesis pathways.ResultsIn this study, the mXcP4H gene from Xanthomonas campestris, as well as the HsAADC, HsAANAT and HIOMT genes from human melatonin synthesis pathway were optimized and introduced into E. coli via a multi-monocistronic vector. The obtained strain BL7992 successfully synthesized 1.13 mg/L melatonin by utilizing L-tryptophan (l-Trp) as a substrate in a shake flask. It was determined that the rate-limiting enzyme for melatonin synthesis is the arylalkylamine N-acetyltransferase, which is encoded by the HsAANAT gene. Targeted metabolomics analysis of l-Trp revealed that the majority of l-Trp flowed to the indole pathway in BL7992, and knockout of the tnaA gene may be beneficial for increasing melatonin production.ConclusionsA metabolic engineering approach was adopted and melatonin was successfully synthesized from low-cost l-Trp in E. coli. This study provides a rapid and economical strategy for the synthesis of melatonin.

The Microbiome in Quiescent Crohn’s Disease With Persistent Symptoms Show Disruptions in Microbial Sulfur and Tryptophan Pathways

Even in the absence of inflammation, persistent symptoms in Crohn's disease (CD) are prevalent and worsen quality of life. Amongst patients without inflammation (quiescent CD), we hypothesized that microbial community structure and function, including tryptophan metabolism, would differ between patients with persistent symptoms (qCD+ S) and without persistent symptoms (qCD-S). We performed a multicenter observational study nested within the Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease. Quiescent inflammation was defined by fecal calprotectin level <150 mcg/g. Persistent symptoms were defined by Crohn's Disease Patient-Reported Outcome-2. Active CD, diarrhea-predominant irritable bowel syndrome, and healthy controls were included as controls. Stool samples underwent whole-genome shotgun metagenomic sequencing. Thirty-nine patients with qCD+ S, 274 qCD-S, 21 active CD, 40 diarrhea-predominant irritable bowel syndrome, and 50 healthy controls were included for analysis. Patients with qCD+ S had a less-diverse microbiome. Furthermore, patients with qCD+ S showed significant enrichment of bacterial species that are normal inhabitants of the oral microbiome (eg Rothia dentocariosa, Fusobacterium nucleatum) and sulfidogenic microbes (eg Prevotella copri, Bilophila spp.). Depletion of important butyrate and indole producers (eg Eubacterium rectale, Faecalibacterium prausnitzii) was also noted in qCD+ S. Potential metagenome-related functional changes in cysteine and methionine metabolism, ATP transport, and redox reactions were disturbed in qCD+ S, also suggestive of altered sulfur metabolism. Finally, qCD+ S showed significant reductions in bacterial tnaA genes, which mediate tryptophan metabolism to indole, and significant tnaA allelic variation compared with qCD-S. The microbiome in qCD+ S showed significant differences in sulfidogenesis, butyrate producers, and typically oral microbes compared to qCD-S and active CD. These results suggest that inflammation may lead to durable microbiome alterations which may mediate persistent symptoms through testable mechanisms.

HicA Toxin-Based Counterselection Marker for Allelic Exchange Mutations in Fusobacterium nucleatum.

The study of fusobacterial virulence factors has dramatically benefited from the creation of various genetic tools for DNA manipulation, including galK-based counterselection for in-frame deletion mutagenesis in Fusobacterium nucleatum, which was recently developed. However, this method requires a host lacking the galK gene, which is an inherent limitation. To circumvent this limitation, we explored the possibility of using the hicA gene that encodes a toxin consisting of a HicAB toxin-antitoxin module in Fusobacterium periodonticum as a new counterselective marker. Interestingly, the full-length hicA gene is not toxic in F. nucleatum, but a truncated hicA gene version lacking the first six amino acids is functional as a toxin. The toxin expression is driven by an rpsJ 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 radD gene within the F. nucleatum rad operon and the tnaA 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 hicA-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. IMPORTANCE 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 hicA gene from Fusobacterium periodonticum expresses an active toxin and was used as a counterselection marker. This hicA-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 hicA gene as a counterselection marker in a bacterial genetic study.

Production of indole and hydrogen sulfide by the oxygen-tolerant mutant strain Clostridium sp. Aeroto-AUH-JLC108 contributes to form a hypoxic microenvironment.

In this study, the oxygen-tolerant mutant strain Clostridium sp. Aeroto-AUH-JLC108 was found to produce indole when grown aerobically. The tnaA gene coding for tryptophanase responsible for the production of indole was cloned. The tnaA gene from Aeroto-AUH-JLC108 is 1677bp and has one point mutation (C36G) compared to the original anaerobic strain AUH-JLC108. Phylogenetic analyses based on the amino acid sequence showed significant homology to that of TnaA from Flavonifractor. Furthermore, we found that the tnaA gene also exhibited cysteine desulfhydrase activity. The production of hydrogen sulfide (H2S) was accompanied by decrease in the amount of the dissolved oxygen in the culture medium. Similarly, the amount of indole produced by strain Aeroto-AUH-JLC108 obviously decreased the oxidation-reduction potential (ORP) in BHI liquid medium. The results demonstrated that production of indole and H2S helped to form a hypoxic microenvironment for strain Aeroto-AUH-JLC108 when grown aerobically.

Construction and Analysis of a Yeast for the Simultaneous Release and Esterification of the Varietal Thiol 3-Sulfanylhexan-1-ol.

Polyfunctional thiols like 3-sulfanylhexan-1-ol (3SH) and its ester 3-sulfanylhexyl acetate (3SHA) are important aroma determinants in wine with exceptionally low odor thresholds. 3SH is largely found in grape must bound to glutathione and cysteine and requires enzymatic action to be perceived sensorially. The wine yeast Saccharomyces cerevisiae is ineffective in releasing volatile thiols from their precursor configuration. For this purpose, a yeast strain was constructed that expresses the carbon-sulfur lyase encoding the tnaA gene from Escherichia coli and overexpresses its native alcohol acetyltransferase encoding genes, ATF1 and ATF2. The resulting yeast strain, which co-expresses tnaA and ATF1, showed elevated 3SH-releasing capabilities and the esterification of 3SH to its acetate ester 3SHA. Levels of over 7000 ng/L of 3SHA in Sauvignon blanc wines were achieved. Enhanced release and esterification of 3SH were also shown in the fermentation of guava and passionfruit pulp and three hop varieties. This study offers prospects for the development of flavor-enhancing yeast strains with optimized thiol-releasing and esterification capabilities in a diverse set of beverage matrices.

RNase E-dependent degradation of tnaA mRNA encoding tryptophanase is prerequisite for the induction of acid resistance in Escherichia coli

Acid-resistance systems are essential for pathogenic Escherichia coli to survive in the strongly acidic environment of the human stomach (pH < 2.5). Among these, the glutamic acid decarboxylase (GAD) system is the most effective. However, the precise mechanism of GAD induction is unknown. We previously reported that a tolC mutant lacking the TolC outer membrane channel was defective in GAD induction. Here, we show that indole, a substrate of TolC-dependent efflux pumps and produced by the tryptophanase encoded by the tnaA gene, negatively regulates GAD expression. GAD expression was restored by deleting tnaA in the tolC mutant; in wild-type E. coli, it was suppressed by adding indole to the growth medium. RNA-sequencing revealed that tnaA mRNA levels drastically decreased upon exposure to moderately acidic conditions (pH 5.5). This decrease was suppressed by RNase E deficiency. Collectively, our results demonstrate that the RNase E-dependent degradation of tnaA mRNA is accelerated upon acid exposure, which decreases intracellular indole concentrations and triggers GAD induction.

Sub-minimum inhibitory concentration ceftazidime inhibits Escherichia coli biofilm formation by influencing the levels of the ibpA gene and extracellular indole

Escherichia coli is a common pathogen of bacterial biofilm infections. Sub-minimum inhibitory concentration ceftazidime (sub-MIC CAZ) could inhibit the biofilm formation of E. coli. Deletion of the ibpAB genes could increase the extracellular indole concentration of E. coli and then inhibit biofilm formation. Therefore, we speculated that sub-MIC CAZ might inhibit biofilm formation via ibpAB. In this study, the results showed that sub-MIC CAZ could significantly inhibit biofilm formation, swimming motility and the expression of the ibpA gene, while it could increase the expression of tnaA gene and extracellular indole concentration. Knockout of the ibpA gene resulted in a decrease in biofilm formation and swimming motility and an increase in the indole concentration. When treated with sub-MIC CAZ, the tnaA gene expression, indole concentration, biofilm formation and swimming motility of MG1655 ΔibpA were similar to those of the control group. The results indicated that sub-MIC CAZ might inhibit the biofilm formation of E. coli by increasing the extracellular indole concentration and downregulating the ibpA gene.

Indole, a Signaling Molecule Produced by the Gut Microbiota, Negatively Impacts Emotional Behaviors in Rats.

Gut microbiota produces a wide and diverse array of metabolites that are an integral part of the host metabolome. The emergence of the gut microbiome-brain axis concept has prompted investigations on the role of gut microbiota dysbioses in the pathophysiology of brain diseases. Specifically, the search for microbe-related metabolomic signatures in human patients and animal models of psychiatric disorders has pointed out the importance of the microbial metabolism of aromatic amino acids. Here, we investigated the effect of indole on brain and behavior in rats. Indole is produced by gut microbiota from tryptophan, through the tryptophanase enzyme encoded by the tnaA gene. First, we mimicked an acute and high overproduction of indole by injecting this compound in the cecum of conventional rats. This treatment led to a dramatic decrease of motor activity. The neurodepressant oxidized derivatives of indole, oxindole and isatin, accumulated in the brain. In addition, increase in eye blinking frequency and in c-Fos protein expression in the dorsal vagal complex denoted a vagus nerve activation. Second, we mimicked a chronic and moderate overproduction of indole by colonizing germ-free rats with the indole-producing bacterial species Escherichia coli. We compared emotional behaviors of these rats with those of germ-free rats colonized with a genetically-engineered counterpart strain unable to produce indole. Rats overproducing indole displayed higher helplessness in the tail suspension test, and enhanced anxiety-like behavior in the novelty, elevated plus maze and open-field tests. Vagus nerve activation was suggested by an increase in eye blinking frequency. However, unlike the conventional rats dosed with a high amount of indole, the motor activity was not altered and neither oxindole nor isatin could be detected in the brain. Further studies are required for a comprehensive understanding of the mechanisms supporting indole effects on emotional behaviors. As our findings suggest that people whose gut microbiota is highly prone to produce indole could be more likely to develop anxiety and mood disorders, we addressed the issue of the inter-individual variability of indole producing potential in humans. An in silico investigation of metagenomic data focused on the tnaA gene products definitively proved this inter-individual variability.

An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications.

Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E.coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500μM, which is within the physiological concentration range occurring during cultivation of E.coli in complex medium. Biotransformations with C43(DE3)_∆tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium.

Physiology and pathogenicity of cpdB deleted mutant of avian pathogenic Escherichia coli

Avian colibacillosis is one of the most common infectious diseases caused partially or entirely by avian pathogenic Escherichia coli (APEC) in birds. In addition to spontaneous infection, APEC can also cause secondary infections that result in greater severity of illness and greater losses to the poultry industry. In order to assess the role of 2′, 3′-cyclic phosphodiesterase (cpdB) in APEC on disease physiology and pathogenicity, an avian pathogenic Escherichia coli-34 (APEC-34) cpdB mutant was obtained using the Red system. The cpdB mutant grew at a slower rate than the natural strain APEC-34. Scanning electron microscopy (SEM) indicated that the bacteria of the cpdB mutant were significantly longer than the bacteria observed in the natural strain (P<0.01), and that the width of the cpdB mutant was significantly smaller than its natural counterpart (P<0.01). In order to evaluate the role of cpdB in APEC in the colonization of internal organs (lung, liver and spleen) in poultry, seven-day-old SPF chicks were infected with 109CFU/chick of the cpdB mutant or the natural strain. No colonizations of cpdB mutants were observed in the internal organs 10days following the infection, though numerous natural strains were observed at 20days following infection. Additionally, the relative expression of division protein ftsZ, outer membrane protein A ompA, ferric uptake regulator fur and tryptophanase tnaA genes in the mutant strain were all significantly lower than in the natural strain (P<0.05 or P<0.01). These results suggested that cpdB is involved in the long-term colonization of APEC in the internal organs of the test subjects. The deletion of the cpdB gene also significantly affected the APEC growth and morphology.

Three Functional Biomarkers for Monitoring the Nanoscale Zero-Valent Iron (nZVI)-Induced Molecular Signature on Soil Organisms

Currently, there are increasing efforts to utilize nanoremediation as an environmental technology for cleaning up polluted environments using nanoscale zero-valent iron (nZVI); however, concerns exist regarding the long-term environmental impact of this strategy. In this study, an innovative methodology for evaluating nZVI impact on soil bacteria is utilized, based on transcriptional analysis of three novel biomakers: tnaA, sodB and trx genes. At the same time, classical toxicological bioassays with the nematode Caenorhabditis elegans were performed. Microcosms treated with 1, 5 and 10 % w/w of nZVI were set up using a commercial standard soil and incubated for 21 days. The tnaA gene, involved in indole production, was significantly upregulated at all assessed nZVI concentrations, suggesting that bacterial cells used this molecule to inform the rest of the community about the changes produced upon nZVI soil treatment. The higher the exposure time, the lower nZVI concentration needed to detect these changes. Consequently, soil bacteria activate a cellular adaptive response to cope with the nZVI-induced oxidative stress, increasing the expression of genes encoding key reactive oxygen species (ROS)-scavenging enzymes; in fact, an upregulation of the sodB and katB genes was recorded upon nZVI exposure. On the contrary, C. elegans survival and growth endpoints were not affected at any nZVI concentration whereas the exposure time significantly increased nematode growth in the soil. Therefore, despite the lack of toxicity revealed by the classical conducted tests, the transcriptional analyses demonstrated the usefulness of combining the set of proposed biomarkers for early detection and monitoring the impact of nZVI on soil bacteria after environmentally important periods of exposure.

Characterization of Tryptophanase from Vibrio cholerae O1

Tryptophanase (Trpase) encoded by the tnaA gene catalyzes the conversion of tryptophan to indole, which is an extracellular signaling molecule detected in various bacteria including Vibrio cholerae. Indole has been demonstrated to regulate biofilm formation, drug resistance, plasmid maintenance and spore formation of bacteria. In the present study, the tnaA gene from V. cholerae O1 (VcTrpase) was cloned and expressed in E. coli BL21(DE3) tn5:tnaA (a Trpase-deficient competent). VcTrpase was purified by Ni2+-NTA chromatography. The obtained VcTrpase had a molecular mass of approximately 49 kDa, a specific activity of 3 U/mg protein, and absorption peaks at 330 and 435nm. Using a site-directed mutagenesis technique, replacement of Arg419 by Val resulted in a VcTrpase completely devoid of activity. Thus, this site can be a target for drug design for controlling V. cholerae.

Characterization of tryptophanase from Vibrio cholerae.

Tryptophanase (Trpase) is a pyridoxal phosphate (PLP)-dependent enzyme responsible for the production of indole, an important intra- and interspecies signaling molecule in bacteria. In this study, the tnaA gene of Vibrio cholerae coding for VcTrpase was cloned into the pET-20b(+) vector and expressed in Escherichia coli BL21(DE3) tn5:tnaA. Using Ni(2+)-nitrilotriacetic acid (NTA) chromatography, VcTrpase was purified, and it possessed a molecular mass of ∼49 kDa with specific absorption peaks at 330 and 435 nm and a specific activity of 3 U/mg protein. The VcTrpase had an 80 % homology to the Trpase of Haemophilus influenzae and E. coli, but only around 50 % identity to the Trpase of Proteus vulgaris and Porphyromonas gingivalis. The optimum conditions for the enzyme were at pH 9.0 and 45 °C. Recombinant VcTrpase exhibited analogous kinetic reactivity to the EcTrpase with K m and k cat values of 0.612 × 10(-3) M and 5.252 s(-1), respectively. The enzyme catalyzed S-methyl-L-cysteine and S-benzyl-L-cysteine degradation, but not L-phenylalanine and L-serine. Using a site-directed mutagenesis technique, eight residues (Thr52, Tyr74, Arg103, Asp137, Arg230, Lys269, Lys270, and His463) were conserved for maintaining enzyme catalysis. All amino acid substitutions at these sites either eliminated or remarkably diminished Trpase activity. These sites are thus potential targets for the design of drugs to control the V. cholerae Trpase and to further investigate its functions.

Development of Indole-3-Acetic Acid-Producing Escherichia coli by Functional Expression of IpdC, AspC, and Iad1

Biosynthesis of indole-3-acetic acid (IAA) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; aminotransferase encoded by aspC, indole-3-pyruvic acid decarboxylase encoded by ipdC, and indole-3-acetic acid dehydrogenase encoded by iad1. The ipdC from Enterobacter cloacae ATCC 13047, aspC from Escherichia coli, and iad1 from Ustilago maydis were cloned and expressed under the control of the tac and sod promoters in E. coli. According to SDS-PAGE and enzyme activity, IpdC and Iad1 showed good expression under the control of P(tac), whereas AspC was efficiently expressed by P(sod) originating from Corynebacterium glutamicum. The activities of IpdC, AspC, and Iad1 from the crude extracts of recombinant E. coli Top 10 were 215.6, 5.7, and 272.1 nmol/min/mg-protein, respectively. The recombinant E. coli DH5α expressing IpdC, AspC, and Iad1 produced about 1.1 g/l of IAA and 0.13 g/l of tryptophol (TOL) after 48 h of cultivation in LB medium with 2 g/l tryptophan. To improve IAA production, a tnaA gene mediating indole formation from tryptophan was deleted. As a result, E. coli IAA68 with expression of the three genes produced 1.8 g/l of IAA, which is a 1.6- fold increase compared with wild-type DH5α harboring the same plasmids. Moreover, the complete conversion of tryptophan to IAA was achieved by E. coli IAA68. Finally, E. coli IAA68 produced 3.0 g/l of IAA after 24 h cultivation in LB medium supplemented with 4 g/l of tryptophan.

New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol

BackgroundGlycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of Escherichia coli physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes.ResultsTranscriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroGfbrtktA, when growing on glycerol, as compared to glucose.ConclusionsThe overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.

Mutations in the Essential Escherichia coli Gene, yqgF, and Their Effects on Transcription

The Escherichia coli yqgF gene is highly conserved across a broad spectrum of bacterial genomes. The gene was first identified as being essential for cell growth during screening for targets for broad-spectrum antibiotics. YqgF is structurally similar to RuvC, a Holliday junction resolvase, but its function has not been established. This study describes the isolation of a temperature-sensitive yqgF mutant, the growth of which was inhibited by rho or nusA multicopy plasmids, indicating that YqgF is involved in transcription. Rho is a global transcription termination factor that acts at Rho-dependent terminator sites, which exist not only at the ends of genes but also within genes. The transcription of genes possessing intragenic, or upstream, Rho-dependent terminators was reduced in temperature-sensitive yqgF mutants. This transcription inhibition was sensitive to the Rho inhibitor, bicyclomycin. In addition, the transcription of mutant tnaA genes defective for upstream Rho-dependent termination was not significantly affected by the yqgF mutation. Taken together, these results suggest that YqgF is involved in anti-termination at Rho-dependent terminators in vivo.

Engineering volatile thiol release in Saccharomyces cerevisiae for improved wine aroma

Volatile thiols, such as 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), are among the most potent aroma compounds found in wine and can have a significant effect on wine quality and consumer preferences. At optimal concentrations in wine, these compounds impart flavours of passionfruit, grapefruit, gooseberry, blackcurrant, lychee, guava and box hedge. The enzymatic release of aromatic thiols from grape-derived, non-volatile cysteinylated precursors (Cys-4MMP and Cys-3MH) and the further modification thereof (conversion of 3MH into 3MHA) during fermentation, enhance the varietal characters of wines such as Sauvignon Blanc. Wine yeast strains have limited and varying capacities to produce aroma-enhancing thiols from their non-volatile counterparts in grape juice. Even under optimal fermentation conditions, the most efficient thiol-releasing Saccharomyces cerevisiae wine strain known realizes less than 5% of the thiol-related flavour potential of grape juice. The objective of this study was to develop a wine yeast able to unleash the untapped thiol aromas in grape juice during winemaking. To achieve this goal, the Escherichia coli tnaA gene, encoding a tryptophanase with strong cysteine-beta-lyase activity, was cloned and overexpressed in a commercial wine yeast strain under the control of the regulatory sequences of the yeast phosphoglycerate kinase I gene (PGK1). This modified strain expressing carbon-sulphur lyase activity released up to 25 times more 4MMP and 3MH in model ferments than the control host strain. Wines produced with the engineered strain displayed an intense passionfruit aroma. This yeast offers the potential to enhance the varietal aromas of wines to predetermined market specifications.

Isolation of an Escherichia coil strain mutant unable to form biofilm on polystyrene and to adhere to human pneumocyte cells: involvement of tryptophanase.

Escherichia coli adherence to biotic and abiotic surfaces constitutes the first step of infection by promoting colonization and biofilm formation. The aim of this study was to gain a better understanding of the relationship between E. coli adherence to different biotic surfaces and biofilm formation on abiotic surfaces. We isolated mutants defective in A549 pneumocyte cells adherence, fibronectin adherence, and biofilm formation by random transposition mutagenesis and sequential passages over A549 cell monolayers. Among the 97 mutants tested, 80 were decreased in biofilm formation, 8 were decreased in A549 cells adherence, 7 were decreased in their adherence to fibronectin, and 17 had no perturbations in either of the three phenotypes. We observed a correlation between adherence to fibronectin or A549 cells and biofilm formation, indicating that biotic adhesive factors are involved in biofilm formation by E. coli. Molecular analysis of the mutants revealed that a transposon insertion in the tnaA gene encoding for tryptophanase was associated with a decrease in both A549 cells adherence and biofilm formation by E. coli. The complementation of the tnaA mutant with plasmid-located wild-type tnaA restored the tryptophanase activity, epithelial cells adherence, and biofilm formation on polystyrene. The possible mechanism of tryptophanase involvement in E. coli adherence and biofilm formation is discussed.

The tryptophanase gene cluster of Haemophilus influenzae type b: evidence for horizontal gene transfer.

Among strains of Haemophilus influenzae, the ability to catabolize tryptophan (as detected by indole production) varies and is correlated with pathogenicity. Tryptophan catabolism is widespread (70 to 75%) among harmless respiratory isolates but is nearly universal (94 to 100%) among strains causing serious disease, including meningitis. As a first step in investigating the relationship between tryptophan catabolism and virulence, we have identified genes in pathogenic H. influenzae which are homologous to the tryptophanase (tna) operon of Escherichia coli. The tna genes are located on a 3.1-kb fragment between nlpD and mutS in the H. influenzae type b (Eagan) genome, are flanked by 43-bp direct repeats of an uptake signal sequence downstream from nlpD, and appear to have been inserted as a mobile unit within this sequence. The organization of this insertion is reminiscent of pathogenicity islands. The tna cluster is found at the same map location in all indole-positive strains of H. influenzae surveyed and is absent from reference type d and e genomes. In contrast to H. influenzae, most other Haemophilus species lack tna genes. Phylogenetic comparisons suggest that the tna cluster was acquired by intergeneric lateral transfer, either by H. influenzae or a recent ancestor, and that E. coli may have acquired its tnaA gene from a related source. Genomes of virulent H. influenzae resemble those of pathogenic enterics in having an island of laterally transferred DNA next to mutS.