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Applied and Environmental Microbiology, March 2008, p. 1717-1725, Vol. 74, No. 6
0099-2240/08/$08.00+0     doi:10.1128/AEM.02589-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cinnamic Acid, an Autoinducer of Its Own Biosynthesis, Is Processed via Hca Enzymes in Photorhabdus luminescens{triangledown}

Sabina Chalabaev,1 Evelyne Turlin,1 Sylvie Bay,2 Christelle Ganneau,2 Emma Brito-Fravallo,1 Jean-François Charles,1 Antoine Danchin,1 and Francis Biville3*

Unité de Génétique des Génomes Bactériens, CNRS URA 2171, Institut Pasteur,1 Unité de Chimie Organique, CNRS URA 2128, Institut Pasteur,2 Unité de Génétique des Membranes Bactériennes, CNRS URA 2172, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France3

Received 16 November 2007/ Accepted 19 January 2008


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ABSTRACT
 
Photorhabdus luminescens, an entomopathogenic bacterium and nematode symbiont, has homologues of the Hca and Mhp enzymes. In Escherichia coli, these enzymes catalyze the degradation of the aromatic compounds 3-phenylpropionate (3PP) and cinnamic acid (CA) and allow the use of 3PP as sole carbon source. P. luminescens is not able to use 3PP and CA as sole carbon sources but can degrade them. Hca dioxygenase is involved in this degradation pathway. P. luminescens synthesizes CA from phenylalanine via a phenylalanine ammonia-lyase (PAL) and degrades it via the not-yet-characterized biosynthetic pathway of 3,5-dihydroxy-4-isopropylstilbene (ST) antibiotic. CA induces its own synthesis by enhancing the expression of the stlA gene that codes for PAL. P. luminescens bacteria release endogenous CA into the medium at the end of exponential growth and then consume it. Hca dioxygenase is involved in the consumption of endogenous CA but is not required for ST production. This suggests that CA is consumed via at least two separate pathways in P. luminescens: the biosynthesis of ST and a pathway involving the Hca and Mhp enzymes.


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INTRODUCTION
 
Cinnamic acid (CA) and its derivatives are secondary metabolites with antioxidant and antibacterial activities produced by plants in response to stressful conditions, such as infections or wounding (21). These compounds are particularly interesting in a wide range of applications. CA is used as a flavoring agent in baked goods, sweets, ice cream, beverages, and chewing gum (33). CA inhibits the growth of several bacteria (7), in particular Escherichia coli O157:H7 (40), and is an active compound in medicinal plants with anti-Helicobacter pylori (1) or antituberculosis activity (2). It also enhances the activity of drugs like isoniazid or rifampin against Mycobacterium tuberculosis (30). CA derivatives also have additional activities: o-methyl cinnamamide inhibits the invasion and metastasis of human malignant melanoma (39), cinnamaldehyde is antimutagenic in mammalian cells (20), and flavonoid compounds with a CA backbone have cancer chemopreventive, antioxidant, and antiasthmatic activities (18). In plants, the synthesis of CA from phenylalanine is catalyzed by phenylalanine ammonia-lyase (PAL; EC 4.3.1.5). In industry, CA is produced by chemical syntheses. As chemical syntheses involve large amounts of solvents, there is a growing interest in developing alternative, environmentally friendly procedures. Nijkamp et al. constructed a Pseudomonas putida strain expressing the PAL enzyme from Rhodosporidium toruloides and secreting CA at a concentration of 5 mM in the medium (27). PAL homologs are found in various plants, fungi, and yeasts but in only two prokaryotes, Streptomyces maritimus (43) and Photorhabdus luminescens (41).

The bacterium P. luminescens (Enterobacteriaceae) is an insect pathogen and a nematode symbiont. After entering the insect host, the nematode, at the infective juvenile stage, releases its bacterial symbionts into the insect hemocoel, in which bacteria proliferate rapidly (10, 13). The insect host succumbs within 24 to 48 h of bacterial infection to bacterial exo- and endotoxins which are produced as the bacteria multiply (3, 4, 17). The bacteria also produce antibiotics that inhibit the growth of competing microorganisms in the insect cadaver (11, 25, 32). CA is a precursor of one of these antibiotics, 3,5-dihydroxy-4-isopropylstilbene (ST). ST is also an inhibitor of melanization, which is a part of the insect immune response (15). The first step of the ST biosynthesis pathway is the deamination of phenylalanine, catalyzed by PAL and resulting in CA (Fig. 1A) (41). The subsequent steps are unknown.


Figure 1
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  		FIG. 1. Utilization of CA by P. luminescens (A) and E. coli (B). (A) Biosynthetic pathway of ST in P. luminescens (41). CA, formed from phenylalanine, is a precursor of ST. (B) Pathway for the degradation of CA by E. coli (12). Metabolites are as follows: cis-3-(3-carboxyethenyl)-3,5-cyclohexadiene-1,2-diol (I), 2,3-dihydroxycinnamic acid (II), 2-hydroxy-6-ketononatrienedioate (III), fumarate (IV), 2-keto-4-pentotenoic acid (V), 4-hydroxy-2-ketovaleric acid (VI), pyruvic acid (VII), acetaldehyde (VIII), and acetyl-CoA (IX). 3PP degradation is catalyzed by the same enzymes.

The P. luminescens strain TT01 genome sequence shows that the bacterium has homologs of the hcaR, -E, -F, -C, -B, and -D genes present in Escherichia coli (14). The E. coli hcaE, -F, -C, -B, and -D operon encodes enzymes involved in the first two steps of CA and 3-phenylpropionate (3PP) catabolism (6, 12) (Fig. 1B). The hca operon and mhp genes allow E. coli to grow on 3PP as sole carbon source (12). Introduction of the E. coli hca and mhp genes into Salmonella enterica serovar Typhimurium allows the growth of S. enterica on CA (12). In addition, hca operon expression in E. coli (12) is positively regulated by HcaR, a member of the LysR family of regulatory proteins (36). This is also the case in P. luminescens (8). In P. luminescens, HcaR is also involved in the oxidative stress response, toxemia, and virulence (8). The effects of HcaR on virulence are not related to its role in the control of hca operon expression, as disruption of hcaE does not affect virulence or toxemia (8).

In the work presented in this report, the involvement of the Hca enzyme in CA utilization and ST synthesis in P. luminescens was investigated.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions.
The P. luminescens strains used in this study were TT01R (a spontaneous rifampin-resistant mutant of P. luminescens subsp. laumondii TT01) (8), used as the wild-type reference in all experiments, its hcaE derivative strain TT01{Delta}hcaE (8), and its hcaR derivative strain TT01{Delta}hcaR (8). P. luminescens strains were routinely grown under aerobic conditions at 30°C in Luria-Bertani (LB) or in Schneider medium (BioWhittaker). If indicated, 3PP (Sigma) and CA (Sigma) were added to a concentration of 1.3 mM each. LB medium was solidified with 1.5% Difco agar as required.

RT-PCR and real-time quantitative RT-PCR.
To determine whether hcaEFCB-plu2208-plu2209 is an operon, reverse transcription-PCR (RT-PCR) was performed using primers designed to amplify three intergenic regions of the hca locus. Strain TT01R was grown in Schneider medium supplemented with 0.5 mM cyclic AMP, in the presence of 1.3 mM 3PP. Bacteria were harvested at the beginning of the stationary phase (optical density at 600 nm of 9). Total RNA was prepared as previously described, using the Trizol method (11). Single-step RT-PCR was performed with 250 ng of total RNA using an AccessQuick RT-PCR system kit (Promega) according to the manufacturer's instructions. The reaction mixture was incubated at 48°C for 45 min and was then subjected to PCR (40 cycles of 30 s at 94°C, 1 min at 55°C, and 2 min at 68°C). We confirmed that none of the RNA samples was contaminated with DNA by performing 40 cycles of PCR with 250 ng of total RNA as the template. The RT-PCR products were analyzed by electrophoresis on 2% agarose gels, using UltraPure agarose (Invitrogen Corp.). Primers were used at a concentration of 1 µM (see Table 1 for primer sequences).


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  		TABLE 1. Nucleotide sequences of primers used in RT-PCR and quantitative RT-PCR experiments

Quantitative RT-PCR was performed as previously described (22). The quantity of cDNA for each gene studied was normalized to the quantity of udp cDNA in each sample; udp was considered to represent a stably expressed housekeeping gene. To check for contaminating chromosomal DNA, each sample was tested in control reactions without reverse transcriptase. The relative change in gene expression was recorded as the ratio of normalized target concentrations (threshold cycle) (26).

PAL enzyme assay.
PAL activity was measured as previously described (23, 41). Bacteria were grown in LB; 40-ml amounts were collected at various times and centrifuged. The cells were resuspended in 2 ml 0.1 M sodium borate buffer (pH 8.8) and sonicated. The protein concentrations in the extracts were determined by using a Bradford reagent assay (Sigma) using bovine serum albumin as the standard. The PAL activity was measured by adding 600 µg of protein into a 20 mM l-phenylalanine solution prepared in 0.1 M sodium borate buffer (pH 8.8). The final reaction mixture volume was 1 ml. The reaction tubes were incubated at 30°C, and the production of CA was detected by measuring the increase in A290.

HPLC analysis.
All samples were filtered through filters with 0.45-µm pores before injection onto the high-pressure liquid chromatography (HPLC) column. 3PP and CA were detected with an Agilent 1100 HPLC system using a Lichrosphere 5 RP-8 column (150 x 4.6 mm). We used the following program: a linear gradient from 0 to 100% acetonitrile in water supplemented with 0.1% trifluoroacetic acid over 20 min; this was followed by the isocratic mode (100% acetonitrile) for 7 min. The mobile phase was delivered at 1 ml/min. 3PP was detected at 210 nm with a 13.31-min retention time. CA was detected at 280 nm with a 13.45-min retention time.

For quantitative analyses, a standard curve of CA was established. The following equation was obtained for those standard solutions with concentrations of CA from 0.013 mM to 1.3 mM: C = 7.200 A – 0.021, where A is the area recorded (recorded units/100,000, with detection at 280 nm) and C is the concentration of CA (mM) in the 20-µl sample injected into the HPLC column. With a total of five points, the coefficient of regression was 0.9981.

Purification and identification of CA.
Strain TT01{Delta}hcaE was grown in 100 ml LB for 48 h. The culture supernatant was dried and eluted through a silica gel column using a solvent made of dichloromethane:methanol (99:1, progressively enriched until 94:6). The presence of putative CA was checked by analyzing elution fractions on a thin-layer chromatography plate (aluminum-backed silica gel 60 F254), using a solvent of dichloromethane:methanol (95:5), which was visualized by UV at 254 nm. Elution fractions containing pure putative CA were then pooled together and evaporated under vacuum. The resulting product was resuspended in dimethyl sulfoxide.

The proton nuclear magnetic resonance (NMR) spectrum of this compound in dimethyl sulfoxide was recorded on a Bruker Avance spectrometer (400 MHz). The analytical results (thin-layer chromatography and NMR) were in agreement with those for commercially available CA.


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RESULTS
 
Photorhabdus luminescens has homologs of genes involved in the known phenylpropionate/cinnamate catabolic pathway.
In E. coli K-12, catabolism of the aromatic compounds 3PP and CA involves the same enzymes (12) (Fig. 1B). The first two steps, catalyzed by the dioxygenase HcaEFCD and the dehydrogenase HcaB, activate the aromatic ring by forming a dihydroxy derivative (compound II in Fig. 1B). Ring cleavage is then catalyzed by MhpB (Fig. 1B). The subsequent steps are catalyzed by other Mhp enzymes, which ultimately break this compound down into Krebs cycle intermediates: fumarate (compound IV), pyruvate (compound VII), and acetyl coenzyme A (acetyl-CoA) (compound IX). The E. coli genes involved in 3PP/CA catabolism are located in two clusters: the hca cluster, encoding proteins involved in the two first steps, and the mhp cluster, encoding proteins involved in the following steps (Fig. 2A). P. luminescens has homologs of the HcaR, -E, -F, -B, -C, and -D proteins, with sequence identities of 47%, 81%, 70%, 62%, and 63%, respectively. The MhpB, MhpC, and MhpD homologs in P. luminescens are Plu2208, Plu2202, and Plu2201, respectively, with amino acid identities of 41% (e-value of 5e-73), 28% (e-value of 8e-20), and 37% (e-value of 2e-49), respectively. Their coding genes flank the hcaR, -E, -F, -C, and -B genes (Fig. 2A). No homolog of mhpE was found. Also, no homolog of mhpF, encoding an acetaldehyde dehydrogenase, was identified, but we did detect an adhE homolog (plu2496), encoding an acetaldehyde dehydrogenase (19). Remarkably, the genes involved in 3PP/CA catabolism are organized into two clusters in E. coli, but their counterparts in P. luminescens all map to a single cluster, with the exception of the hcaT homolog (putative transporter of 3PP), located outside the cluster. The hcaR gene, which presumably encodes the regulator of the putative operon, is transcribed divergently, as is the case for many LysR-type regulators (34) (Fig. 2A).


Figure 2
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  		FIG. 2. (A) Genetic organization of the hca and mhp genes in the E. coli and P. luminescens chromosomes. Solid arrows indicate the direction of gene transcription. Dotted arrows indicate homology between plu2201 and mhpD, plu2202 and mhpC, and plu2208 and mhpB. For hca genes, genes with similar shadings encode subunits of the same protein. (B) Detection of the hcaEFCB-plu2208-plu2209 transcript by RT-PCR. RT-PCR products were analyzed by electrophoresis in 2% agarose gels. RT-PCR was performed with (+) and without (–) reverse transcriptase. Lanes 1 and 2, hcaEFCB transcript (expected size, 2.5 kb), produced with primers RThcaE1 and RThcaB2; lanes 3 and 4, hcaB-plu2208 transcript (expected size, 1.4 kb), produced with primers RThcaB1 and RTplu2208_2; lanes 5 and 6, plu2208-plu2209 transcript (expected size, 1.6 kb), produced with primers RTplu2208_1 and RTplu2209_2 (see Table 1 for primer sequences); lane M, SmartLadder size marker. Molecular sizes are indicated on the left.

The hca operon of P. luminescens includes an additional gene, plu2208, not found in the E. coli operon (Fig. 2A). Plu2208 is a homolog of MhpB, catalyzing the third step of 3PP/CA catabolism in E. coli (Fig. 1B). This led us to investigate whether hcaE, hcaF, hcaC, plu2208 (mhpB homolog), and plu2209 (hcaD homolog) were expressed as a single operon in P. luminescens. RT-PCRs were carried out with three pairs of primers (Table 1; Fig. 2B). Total RNA was extracted from the TT01R strain grown in Schneider medium supplemented with 1.3 mM 3PP to induce the expression of the putative hca operon and cyclic AMP to prevent the glucose-dependent repression of hca gene expression (38). An amplicon of the expected size was obtained in all cases: 2.5 kb for the hcaEFCB amplicon, 1.4 kb for the hcaB-plu2208 amplicon, and 1.6 kb for the plu2208-plu2209 amplicon (Fig. 2B). Thus, in P. luminescens, hcaE, -F, -C, and -B and plu2208 and plu2209 are expressed as an operon, whose homologs in E. coli are involved in the first three steps of 3PP/CA catabolism and result in the ring-cleavage product of 3PP/CA.

Photorhabdus luminescens is not able to use 3PP or CA as a sole carbon source, but degrades them.
The hca and mhp genes allow E. coli to grow on 3PP as the sole carbon source (6, 12). Whether this was also the case in P. luminescens was investigated. Bacteria TT01R did not grow in minimal medium containing 3PP. The introduction of the E. coli hca and mhp genes into Salmonella enterica serovar Typhimurium allows S. enterica growth on CA (12). The ability of P. luminescens to grow in minimal medium containing CA as the sole carbon source was tested. No growth was observed. This inability may be related to the fact that no mhpE and mhpF homologs, required for the two last steps of catabolism, were identified. However, E. coli, despite the presence of mhpE and mhpF, degrades CA but does not use it as sole carbon source (12).

The degradation of 3PP or CA by P. luminescens was investigated. The wild-type strain TT01R was grown in LB medium supplemented with 1.3 mM 3PP or 1.3 mM CA, and the 3PP and CA amounts in the culture supernatants were assayed by HPLC analysis. Both the 3PP and CA quantities rapidly decreased between 16 and 24 h of culture, during the transition from exponential growth to stationary phase (Fig. 3). The involvement of Hca enzymes in this process was investigated; thus, the degradation of 3PP and CA by the TT01{Delta}hcaE mutant strain grown in the presence of these compounds was monitored. The first gene of the hca operon, hcaE, encoding the alpha subunit of the Hca dioxygenase which catalyzes the first step of 3PP/CA catabolism in E. coli, was disrupted in this mutant (Fig. 1B). The amounts of 3PP and CA in culture supernatants of strain TT01{Delta}hcaE remained constant during growth from 0 to 80 h (Fig. 3B). Thus, the consumption of 3PP and CA requires the activity of the Hca dioxygenase. This result establishes that the first step of 3PP/CA degradation catalyzed by the Hca dioxygenase occurs in P. luminescens.


Figure 3
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  		FIG. 3. Consumption of 3PP and CA by P. luminescens. The parent TT01R and mutant TT01{Delta}hcaE strains were grown in LB medium containing 1.3 mM 3PP or 1.3 mM CA. Culture supernatants were collected at various times and analyzed by HPLC. (A) Chromatograms of culture supernatants of TT01R, recorded with detection at 210 nm for 3PP-containing culture and 280 nm for CA-containing supernatants. (B) 3PP (open symbols) and CA (closed symbols) quantities remaining in culture supernatants, and cell densities (dotted line) of TT01R and TT01{Delta}hcaE. Quantities of 3PP and CA are expressed as percentage of area of corresponding peak compared with initial area. O.D.600, optical density at 600 nm.

HcaE is involved in degradation of endogenous CA.
Unlike most known enterobacteria, P. luminescens expresses a PAL enzyme, catalyzing CA formation from phenylalanine (41). CA was previously detected in bacterial extracts (5). The presence of CA in the culture supernatants of TT01R grown in LB medium was analyzed by HPLC. A compound was secreted by P. luminescens that accumulated after 16 h of growth (Fig. 4A). This compound eluted at the same position as CA, and it had a similar UV spectrum to CA (data not shown). After purification on silica gel, the identity of this compound was confirmed by proton NMR (data not shown). The concentration of CA released by TT01R into the culture supernatant increased at the end of the exponential growth phase, between 8 and 16 h of culture, reaching 0.2 mM, and then it rapidly decreased to 0.05 mM after 20 h (Fig. 4B). The CA concentration eventually stabilized at 0.01 mM (Fig. 4B). Thus, P. luminescens released a considerable amount of CA into the medium and then subsequently processed it.


Figure 4
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  		FIG. 4. Detection of CA in culture supernatants of P. luminescens. (A) HPLC chromatograms of LB medium (top) and of a 16-h-culture supernatant of TT01R grown in LB medium (bottom). Detection was performed at 280 nm. (B) The parent TT01R (diamonds) and the mutant TT01{Delta}hcaE (triangles) bacteria were grown in LB. Concentrations of CA in culture supernatant were determined by HPLC. Measurements are expressed as the means ± standard deviations of the results of two independent experiments. O.D.600, optical density at 600 nm.

HcaE is involved in the degradation of exogenous CA. To know whether HcaE is involved in the degradation of endogenous CA, the culture supernatant of strain TT01{Delta}hcaE grown in LB was analyzed by HPLC. The concentration of CA increased at the end of the exponential growth phase, between 8 and 16 h of growth (Fig. 4B). After 16 h, in contrast to the results for the parental strain TT01R, the CA concentration kept increasing, reaching a value of 1.5 mM after 4 days of growth (Fig. 4B). Thus, the disruption of hcaE abolished the stationary-phase-dependent decrease of extracellular CA evidenced in the wild-type strain. This suggests that the Hca dioxygenase is involved in the catabolism of endogenous CA from TT01R culture supernatants.

Effect of hcaE disruption on ST production.
In P. luminescens, CA is a precursor of the antibiotic ST (41). The pathway responsible for ST formation from CA is still unknown. Hca dioxygenase is involved in the consumption of exogenous and endogenous CA. As a consequence, Hca dioxygenase may catalyze the first step of the CA-to-ST pathway. The overlay method was used to test whether HcaE is required for ST formation (41). TT01R and TT01{Delta}hcaE mutant bacteria were spotted on LB agar and overlaid with soft agar containing Micrococcus luteus bacteria. The growth of M. luteus was inhibited around the P. luminescens spot, this inhibition resulting from ST antibiotic activity (41). Greater ST production by P. luminescens results in a larger inhibition zone. The diameter of the inhibition zone was 2.8 ± 0.1 (mean ± standard deviation) cm for the parent and 3.3 ± 0.1 cm for the mutant (n = 2), indicating that HcaE is not required for ST production.

Effect of hcaE disruption on PAL activity and stlA expression.
The increase in the amount of extracellular endogenous CA resulting from hcaE disruption may be related only to a decrease in CA degradation, but it also may be the consequence of an increase in CA synthesis. Thus, the effect of hcaE disruption on PAL activity was investigated. Strains TT01R and TT01{Delta}hcaE were grown in LB; proteins were extracted at various times, and the PAL activity was measured. Interestingly, the PAL activity measured in crude bacterial extract was about threefold higher in the mutant than in the parent strain (Fig. 5A). The activity measured between 14 and 44 h of growth was 160 ± 70 pmol min–1 mg–1 in the TT01R extracts and 430 ± 100 pmol min–1 mg–1 in the TT01{Delta}hcaE extracts (Fig. 5A). An increase of PAL activity in the hcaE mutant may be due to an increase in the expression of the stlA gene, coding for the PAL enzyme. The stlA mRNA amount was measured by quantitative RT-PCR in total RNA extracted from strains TT01R and TT01{Delta}hcaE grown in LB medium. The expression of stlA was higher in the mutant than in the parent (Fig. 5B). This shows that hcaE disruption increases stlA expression, thus enhancing CA synthesis.


Figure 5
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  		FIG. 5. CA synthesis by P. luminescens. The parent TT01R and the mutant TT01{Delta}hcaE strain were grown in LB. (A) Proteins were extracted at various times of growth, and PAL activity was measured. (B) Total RNA was extracted at various times of growth, and amount of stlA mRNA was measured by quantitative RT-PCR, using expression of udp for normalization. Expression levels of stlA in TT01R after 16 h of growth were arbitrarily fixed to 1. Measurements are expressed as the means ± standard deviation of the results of two independent experiments.

CA induces its own biosynthesis.
The increase in PAL activity resulting from hcaE disruption may be the consequence of an increase in the amount of endogenous CA. Indeed, CA could induce its own synthesis similarly to other compounds, such as 2,4-diacetylphloroglucinol (an antibacterial phenolic compound) (35). The effect of CA on PAL activity was investigated. The wild-type strain TT01R was grown in LB in the presence or absence of 1.3 mM exogenous CA, and proteins were extracted at various times. The presence of exogenous CA strongly increased PAL activity—28-fold—after 19 h of growth (Fig. 6A). The increase occurred only after 19 h of growth, coinciding with the disappearance of exogenous CA from the extracellular medium (Fig. 3B). This increase may be related to the internalization of exogenous CA, which may start when CA begins to disappear from the extracellular medium.


Figure 6
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  		FIG. 6. Autoinduction of CA synthesis. (A) Effect of CA on PAL activity. TT01R was grown in LB with or without 1.3 mM CA. Proteins were extracted at various times, PAL activity was measured, and the increase factor was determined. The increase factor is expressed as the ratio of activity measured in the presence of exogenous CA over activity in the absence of exogenous CA. (B) Effect of CA on stlA expression. The parent strain TT01R and the mutant strain TT01{Delta}hcaR were grown in LB with or without 1.3 mM CA. Total RNA was extracted at various times of growth, and the amount of stlA mRNA was measured by quantitative RT-PCR, using the expression of udp for normalization. The induction factor is expressed as the ratio of the amount measured in the presence of exogenous CA over the amount in the absence of exogenous CA. Measurements are expressed as the means ± standard deviation of the results of two independent experiments.

This increase in PAL activity may be the consequence of inducing the expression of its structural gene, stlA. The amount of stlA mRNA was measured by quantitative RT-PCR in TT01R bacteria grown in LB in the presence or absence of 1.3 mM CA. After 17 h of growth, the amount of stlA mRNA was 5.1 ± 1.7 times greater in bacteria grown in the presence than in the absence of exogenous CA (n = 2) (Fig. 6B). After 20 and 26 h of growth, stlA expression was similar in bacteria grown with or without exogenous CA (Fig. 6B). Thus, the increase in PAL activity by CA was due to the induction of stlA expression.

Involvement of the HcaR regulator in CA-induced hcaE and stlA expression.
In E. coli, CA induces the expression of the hca operon, and this induction requires the HcaR regulatory protein (12). The effect of CA on hcaE expression in P. luminescens was assessed by quantitative RT-PCR. In TT01R grown without exogenous CA, hcaE was expressed at the end of the exponential growth (Fig. 7). The presence of exogenous CA increased hcaE expression by 6.3 ± 2.5 times after 15 h (Fig. 7). Thus, CA induces the expression of the hca operon in P. luminescens. In the TT01{Delta}hcaR mutant bacteria, disrupted for hcaR, this induction did not occur (Fig. 7). Thus, in P. luminescens, HcaR is required for CA-induced hcaE expression during the stationary phase.


Figure 7
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  		FIG. 7. Effect of CA on expression of hcaE, plu2201, and plu2202. The parent TT01R and the mutant TT01{Delta}hcaR strains were grown in LB with (dotted lines) or without (solid lines) 1.3 mM CA. Total RNA was extracted at different times of growth, and amounts of hcaE, plu2201, and plu2202 mRNA were measured by quantitative RT-PCR, using expression of udp for normalization. The expression level in TT01R bacteria grown for 15 h in the absence of exogenous CA was arbitrarily fixed at 1. Measurements are expressed as the means ± standard deviations of the results of two independent experiments.

In P. luminescens, the hca operon encodes homologs of various enzymes that catalyze the first three steps of CA degradation (Fig. 1B and 2). plu2201 and plu2202 are located upstream of the hcaR gene and encode homologs of enzymes that catalyze the fourth and the fifth steps of CA degradation (Fig. 1B and 2). We investigated by quantitative RT-PCR whether plu2201 and plu2202 expression is induced by CA. In the parent TT01R strain, the expression patterns of plu2201 and plu2202 are similar to that of hcaE (Fig. 7). After 15 h of growth, plu2201 expression was 5.1 ± 0.8 times greater and plu2202 expression was 5.7 ± 0.1 times greater in the presence of exogenous CA than in its absence (Fig. 7). This showed that CA also induces plu2201 and plu2202 expression. No induction occurred in the TT01{Delta}hcaR mutant strain (Fig. 7). Therefore, the effect of CA on plu2201 and plu2202 expression requires HcaR.

CA induces the expression of the hca operon and the plu2201 and plu2202 genes. It also induces stlA expression. To know whether HcaR was required for this latter induction effect, stlA expression was measured in the TT01{Delta}hcaR mutant strain grown with or without 1.3 mM CA in LB medium. The presence of exogenous CA increased the amount of stlA mRNA 2.2 times in the TT01{Delta}hcaR mutant after 17 h of growth (Fig. 6B). This shows that HcaR is not required for CA-induced stlA expression.


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DISCUSSION
 
Photorhabdus luminescens is one of the two known bacterial species able to synthesize CA from phenylalanine (Fig. 1A) (41). CA is the precursor for synthesizing ST, which has antibiotic activity (15, 32, 41). P. luminescens also produces homologs of the Hca enzymes catalyzing CA degradation in E. coli (Fig. 1B) (12). In this study, Hca enzymes from P. luminescens are shown to be involved in the degradation of endogenous CA, but not required for ST synthesis. CA was processed via at least two distinct pathways in P. luminescens.

P. luminescens releases CA at the end of exponential growth, with its concentration reaching 0.2 mM in the culture supernatant, and then rapidly degrades it at the beginning of stationary phase. The Hca dioxygenase is involved in this degradation. The first steps of lincomycin A biosynthesis in Streptomyces lincolnensis comprise the oxidation of the aromatic ring of tyrosine by dioxygenases (28). Thus, the Hca dioxygenase could catalyze the first step of the ST biosynthetic pathway in P. luminescens. However, HcaE, the alpha subunit of the Hca dioxygenase, was not required for ST production, strongly suggesting that the Hca dioxygenase is not required for ST synthesis. Besides, the ring-cleavage product of CA (compound III in Fig. 1) is probably not an intermediary compound of ST formation, strongly suggesting that the Hca enzymes are not involved in the ST pathway.

The maximal concentration of ST released by P. luminescens when grown in LB was 0.03 mM during the stationary phase (data not shown), while 0.2 mM CA was consumed. Thus, ST is a minority final product of CA processing. It means that either (1) CA is predominantly processed via the pathway involving the Hca enzymes or (2) ST is just an intermediate compound. The latter is plausible, as stilbene can react in many different ways, forming a variety of stilbene dimers and trimers or different stilbene glucosides in seemingly endless combinations (9, 24). The expression pattern of the hca operon coincided with the disappearance of CA from the culture supernatant. In addition, this disappearance was abolished by hcaE disruption. Together, these results suggest that the Hca dioxygenase is responsible for the majority of CA degradation.

In E. coli, the CA ring-cleavage product is eventually degraded into Krebs cycle intermediates by Mhp enzymes (12) (Fig. 1B). The P. luminescens homologs of mhpC and mhpD, plu2202 and plu2201, respectively, have an expression pattern similar to that of hcaE. Thus, the first five reactions of CA catabolism do occur in P. luminescens. No mhpE or mhpF homologs were found in the genome of P. luminescens, which is unable to use 3PP or CA as the sole carbon source. These observations suggest that the Krebs cycle intermediates pyruvate and acetyl-CoA are not the final products of CA degradation in P. luminescens. The role of the Hca enzymes in the P. luminescens life cycle remains to be understood. The Hca dioxygenase is not involved in pathogenicity, as the disruption of hcaE does not affect P. luminescens virulence in Bombyx mori larvae (8). A P. luminescens mutant disrupted for stlA, which does not produce CA, has a delayed virulence in Manduca sexta larvae (15). However, this delay was shown to be due to the absence of ST production, and not an absence of CA production (15).

CA induces the expression of genes involved in its synthesis (stlA) via an unknown regulator and in its degradation (the hca operon, plu2201, and plu2202) via the HcaR regulatory protein. CA synthesis self-induction may explain why the disruption of hcaE increases stlA expression. The disruption of hcaE increased the amount of intracellular CA (data not shown), which may in turn increase stlA expression, leading to increases in PAL activity and ST production. Other examples of autoinduction in bacteria have been reported, such as the behavior of siderophore pyochelin in Pseudomonas aeruginosa (31) or yersiniabactin in Yersinia enterocolitica (29). The best-known autoinducers are quorum-sensing molecules, like N-acyl-homoserine lactones (16). In P. luminescens, the regulation of the CA level shares common features with that of a quorum-sensing molecule, autoinducer 2 (AI-2). E. coli and S. enterica serovar Typhimurium release AI-2 into the medium at the end of exponential growth and then internalize it at the start of the stationary phase, making AI-2 disappear from the culture supernatant (37, 42). After being internalized, AI-2 is converted to currently uncharacterized products. A similar behavior for CA in P. luminescens is reported here. Besides, both AI-2 and CA are small molecules with similar molecular sizes (150 and 148 g mol–1, respectively), and both are able to activate genetic expression. Therefore, it should be interesting to investigate whether CA itself is a quorum-sensing molecule.

In conclusion, in P. luminescens, CA induces its own synthesis, is actively released into the medium at the end of exponential growth, and is then itself consumed by at least two pathways: the ST biosynthetic pathway and a pathway involving the Hca enzymes, yet to be characterized (Fig. 8). The parallels established between AI-2 and CA may provide some direction to future studies.


Figure 8
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  		FIG. 8. Proposed model for CA utilization and response in P. luminescens. PAL converts phenylalanine to CA. CA is then processed via the ST biosynthetic pathway (encircled question mark) and via a catabolic pathway involving homologs of the Hca and Mhp enzymes. Final products (bold question mark) of the latter pathway are unknown. CA also induces its own synthesis by inducing the expression of stlA, coding for PAL via an unknown regulator (encircled question mark). CA also induces its catabolism by inducing the expression of the hca operon and the plu2201 and plu2202 genes, coding for the Hca and Mhp homologs. This induction requires the HcaR regulator.


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ACKNOWLEDGMENTS
 
We are grateful to Frédéric Bonhomme for performing NMR spectrometry analyses, to John M. Webster and Bin Li for providing ST, and to Marie-Françoise Hullo for technical assistance.

Financial support was provided by the Institut Pasteur, the Centre National de la Recherche Scientifique (URA 2171, URA 2172, and URA 2128), the Probactys European NEST program (CT-2006-029104), and the Pasteur-Weizmann Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Unité de Génétique des Membranes Bactériennes, CNRS URA 2172, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: 33 1 40 61 32 77. Fax: 33 1 45 68 89 38. E-mail: fbiville{at}pasteur.fr Back

{triangledown} Published ahead of print on 1 February 2008. Back


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REFERENCES
 
    1
  1. Bae, E. A., M. J. Han, N. J. Kim, and D. H. Kim. 1998. Anti-Helicobacter pylori activity of herbal medicines. Biol. Pharm. Bull. 21:990-992.[Medline]
    2. 2
  2. Barnes, C. C., M. K. Smalley, K. P. Manfredi, K. Kindscher, H. Loring, and D. M. Sheeley. 2003. Characterization of an anti-tuberculosis resin glycoside from the prairie medicinal plant Ipomoea leptophylla. J. Nat. Prod. 66:1457-1462.[CrossRef][Medline]
    3. 3
  3. Bowen, D., T. A. Rocheleau, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia, and R. H. ffrench-Constant. 1998. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280:2129-2132.[Abstract/Free Full Text]
    4. 4
  4. Bowen, D. J., and J. C. Ensign. 1998. Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl. Environ. Microbiol. 64:3029-3035.[Abstract/Free Full Text]
    5. 5
  5. Brachmann, A. O., S. A. Joyce, H. Jenke-Kodama, G. Schwar, D. J. Clarke, and H. B. Bode. 2007. A type II polyketide synthase is responsible for anthraquinone biosynthesis in Photorhabdus luminescens. Chembiochem 8:1721-1728.[CrossRef][Medline]
    6. 6
  6. Burlingame, R. P., and P. J. Chapman. 1983. Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli. J. Bacteriol. 155:113-121.[Abstract/Free Full Text]
    7. 7
  7. Burt, S. 2004. Essential oils: their antibacterial properties and potential applications in foods—a review. Int. J. Food Microbiol. 94:223-253.[CrossRef][Medline]
    8. 8
  8. Chalabaev, S., E. Turlin, J.-F. Charles, A. Namane, S. Pagès, A. Givaudan, E. Brito-Fravallo, A. Danchin, and F. Biville. 2007. The HcaR regulatory protein of Photorhabdus luminescens affects the production of proteins involved in oxidative stress and toxemia. Proteomics 7:4499-4510.[CrossRef][Medline]
    9. 9
  9. Cichewicz, R. H., S. A. Kouzi, and M. T. Hamann. 2000. Dimerization of resveratrol by the grapevine pathogen Botrytis cinerea. J. Nat. Prod. 63:29-33.[CrossRef][Medline]
    10. 10
  10. Daborn, P. J., N. Waterfield, M. A. Blight, and R. H. ffrench-Constant. 2001. Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection. J. Bacteriol. 183:5834-5839.[Abstract/Free Full Text]
    11. 11
  11. Derzelle, S., E. Duchaud, F. Kunst, A. Danchin, and P. Bertin. 2002. Identification, characterization, and regulation of a cluster of genes involved in carbapenem biosynthesis in Photorhabdus luminescens. Appl. Environ. Microbiol. 68:3780-3789.[Abstract/Free Full Text]
    12. 12
  12. Diaz, E., A. Ferrandez, and J. L. Garcia. 1998. Characterization of the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid in Escherichia coli K-12. J. Bacteriol. 180:2915-2923.[Abstract/Free Full Text]
    13. 13
  13. Dowds, B. C. A., and A. Peters. 2002. Virulence mechanism, p. 79-98. In R. Gaugler (ed.), Entomopathogenic nematology. CABI Publishing, Wallingford, United Kingdom.
    14. 14
  14. Duchaud, E., C. Rusniok, L. Frangeul, C. Buchrieser, A. Givaudan, S. Taourit, S. Bocs, C. Boursaux-Eude, M. Chandler, J. F. Charles, E. Dassa, R. Derose, S. Derzelle, G. Freyssinet, S. Gaudriault, C. Medigue, A. Lanois, K. Powell, P. Siguier, R. Vincent, V. Wingate, M. Zouine, P. Glaser, N. Boemare, A. Danchin, and F. Kunst. 2003. The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens. Nat. Biotechnol. 21:1307-1313.[CrossRef][Medline]
    15. 15
  15. Eleftherianos, I., S. Boundy, S. A. Joyce, S. Aslam, J. W. Marshall, R. J. Cox, T. J. Simpson, D. J. Clarke, R. H. ffrench-Constant, and S. E. Reynolds. 2007. An antibiotic produced by an insect-pathogenic bacterium suppresses host defenses through phenoloxidase inhibition. Proc. Natl. Acad. Sci. USA 104:2419-2424.[Abstract/Free Full Text]
    16. 16
  16. Fuqua, C., and E. P. Greenberg. 1998. Self perception in bacteria: quorum sensing with acylated homoserine lactones. Curr. Opin. Microbiol. 1:183-189.[CrossRef][Medline]
    17. 17
  17. Guo, L., R. O. Fatig III, G. L. Orr, B. W. Schafer, J. A. Strickland, K. Sukhapinda, A. T. Woodsworth, and J. K. Petell. 1999. Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins. Purification and characterization of toxin A and toxin B. J. Biol. Chem. 274:9836-9842.[Abstract/Free Full Text]
    18. 18
  18. Kaneko, M., E. I. Hwang, Y. Ohnishi, and S. Horinouchi. 2003. Heterologous production of flavanones in Escherichia coli: potential for combinatorial biosynthesis of flavonoids in bacteria. J. Ind. Microbiol. Biotechnol. 30:456-461.[CrossRef][Medline]
    19. 19
  19. Kessler, D., I. Leibrecht, and J. Knappe. 1991. Pyruvate-formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE. FEBS Lett. 281:59-63.[CrossRef][Medline]
    20. 20
  20. King, A. A., D. T. Shaughnessy, K. Mure, J. Leszczynska, W. O. Ward, D. M. Umbach, Z. Xu, D. Ducharme, J. A. Taylor, D. M. Demarini, and C. B. Klein. 2007. Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair. Mutat. Res. 616:60-69.[Medline]
    21. 21
  21. Korkina, L. G. 2007. Phenylpropanoids as naturally occurring antioxidants: from plant defense to human health. Cell. Mol. Biol. (Noisy-Le-Grand) 53:15-25.[Medline]
    22. 22
  22. Krin, E., N. Chakroun, E. Turlin, A. Givaudan, F. Gaboriau, I. Bonne, J. C. Rousselle, L. Frangeul, C. Lacroix, M. F. Hullo, L. Marisa, A. Danchin, and S. Derzelle. 2006. Pleiotropic role of quorum-sensing autoinducer 2 in Photorhabdus luminescens. Appl. Environ. Microbiol. 72:6439-6451.[Abstract/Free Full Text]
    23. 23
  23. Kyndt, J. A., T. E. Meyer, M. A. Cusanovich, and J. J. Van Beeumen. 2002. Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein. FEBS Lett. 512:240-244.[CrossRef][Medline]
    24. 24
  24. Langcake, P., and R. J. Pryce. 1977. A new class of phytoalexins from grapevines. Experientia 33:151-152.[CrossRef][Medline]
    25. 25
  25. Li, J., G. Chen, H. Wu, and J. M. Webster. 1995. Identification of two pigments and a hydroxystilbene antibiotic from Photorhabdus luminescens. Appl. Environ. Microbiol. 61:4329-4333.[Abstract]
    26. 26
  26. Livak, K., and T. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402-408.[CrossRef][Medline]
    27. 27
  27. Nijkamp, K., N. van Luijk, J. A. de Bont, and J. Wery. 2005. The solvent-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose. Appl. Microbiol. Biotechnol. 69:170-177.[CrossRef][Medline]
    28. 28
  28. Novotna, J., A. Honzatko, P. Bednar, J. Kopecky, J. Janata, and J. Spizek. 2004. l-3,4-Dihydroxyphenyl alanine-extradiol cleavage is followed by intramolecular cyclization in lincomycin biosynthesis. Eur. J. Biochem. 271:3678-3683.[Medline]
    29. 29
  29. Pelludat, C., A. Rakin, C. A. Jacobi, S. Schubert, and J. Heesemann. 1998. The yersiniabactin biosynthetic gene cluster of Yersinia enterocolitica: organization and siderophore-dependent regulation. J. Bacteriol. 180:538-546.[Abstract/Free Full Text]
    30. 30
  30. Rastogi, N., K. S. Goh, L. Horgen, and W. W. Barrow. 1998. Synergistic activities of antituberculous drugs with cerulenin and trans-cinnamic acid against Mycobacterium tuberculosis. FEMS Immunol. Med. Microbiol. 21:149-157.[CrossRef][Medline]
    31. 31
  31. Reimmann, C., L. Serino, M. Beyeler, and D. Haas. 1998. Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF genes, are induced by extracellular pyochelin in Pseudomonas aeruginosa. Microbiology 144:3135-3148.[Abstract/Free Full Text]
    32. 32
  32. Richardson, W. H., T. M. Schmidt, and K. H. Nealson. 1988. Identification of an anthraquinone pigment and a hydroxystilbene antibiotic from Xenorhabdus luminescens. Appl. Environ, Microbiol. 54:1602-1605.[Abstract/Free Full Text]
    33. 33
  33. Roller, S., and P. Seedhar. 2002. Carvacrol and cinnamic acid inhibit microbial growth in fresh-cut melon and kiwifruit at 4 degrees and 8 degrees C. Lett. Appl. Microbiol. 35:390-394.[CrossRef][Medline]
    34. 34
  34. Schell, M. A. 1993. Molecular biology of the LysR family of transcriptional regulators. Annu. Rev. Microbiol. 47:597-626.[CrossRef][Medline]
    35. 35
  35. Schnider-Keel, U., A. Seematter, M. Maurhofer, C. Blumer, B. Duffy, C. Gigot-Bonnefoy, C. Reimmann, R. Notz, G. Defago, D. Haas, and C. Keel. 2000. Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin. J. Bacteriol. 182:1215-1225.[Abstract/Free Full Text]
    36. 36
  36. Stragier, P., F. Richaud, F. Borne, and J. C. Patte. 1983. Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli. I. Identification of a lysR gene encoding an activator of the lysA gene. J. Mol. Biol. 168:307-320.[CrossRef][Medline]
    37. 37
  37. Taga, M. E., S. T. Miller, and B. L. Bassler. 2003. Lsr-mediated transport and processing of AI-2 in Salmonella typhimurium. Mol. Microbiol. 50:1411-1427.[CrossRef][Medline]
    38. 38
  38. Turlin, E., M. Perrotte-Piquemal, A. Danchin, and F. Biville. 2001. Regulation of the early steps of 3-phenylpropionate catabolism in Escherichia coli. J. Mol. Microbiol. Biotechnol. 3:127-133.[Medline]
    39. 39
  39. Welch, D. R., D. E. Harper, and K. H. Yohem. 1993. U-77,863: a novel cinnanamide isolated from Streptomyces griseoluteus that inhibits cancer invasion and metastasis. Clin. Exp. Metastasis 11:201-212.[CrossRef][Medline]
    40. 40
  40. Wells, J. E., E. D. Berry, and V. H. Varel. 2005. Effects of common forage phenolic acids on Escherichia coli O157:H7 viability in bovine feces. Appl. Environ. Microbiol. 71:7974-7979.[Abstract/Free Full Text]
    41. 41
  41. Williams, J. S., M. Thomas, and D. J. Clarke. 2005. The gene stlA encodes a phenylalanine ammonia-lyase that is involved in the production of a stilbene antibiotic in Photorhabdus luminescens TT01. Microbiology 151:2543-2550.[Abstract/Free Full Text]
    42. 42
  42. Xavier, K. B., and B. L. Bassler. 2005. Regulation of uptake and processing of the quorum-sensing autoinducer AI-2 in Escherichia coli. J. Bacteriol. 187:238-248.[Abstract/Free Full Text]
    43. 43
  43. Xiang, L., and B. S. Moore. 2002. Inactivation, complementation, and heterologous expression of encP, a novel bacterial phenylalanine ammonia-lyase gene. J. Biol. Chem. 277:32505-32509.[Abstract/Free Full Text]

Applied and Environmental Microbiology, March 2008, p. 1717-1725, Vol. 74, No. 6
0099-2240/08/$08.00+0     doi:10.1128/AEM.02589-07
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