APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2002, p. 3780–3789 0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.8.3780–3789.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 68, No. 8
Identification, Characterization, and Regulation of a Cluster of Genes Involved in Carbapenem Biosynthesis in Photorhabdus luminescens Sylviane Derzelle,1* Eric Duchaud,2 Frank Kunst,2 Antoine Danchin,1,3 and Philippe Bertin1† Unite´ de Ge´ne´tique des Ge´nomes Bacte´riens1 and Laboratoire de Ge´nomique des Microorganismes Pathoge`nes,2 Institut Pasteur, 75724 Paris Cedex 15, France, and HKU-Pasteur Research Centre, Pokfulam, Hong Kong3 Received 25 January 2002/Accepted 29 May 2002
The luminescent entomopathogenic bacterium Photorhabdus luminescens produces several yet-uncharacterized broad-spectrum antibiotics. We report the identification and characterization of a cluster of eight genes (named cpmA to cpmH) responsible for the production of a carbapenem-like antibiotic in strain TT01 of P. luminescens. The cpm cluster differs in several crucial aspects from other car operons. The level of cpm mRNA peaks during exponential phase and is regulated by a Rap/Hor homolog identified in the P. luminescens genome. Marker-exchange mutagenesis of this gene in the entomopathogen decreased antibiotic production. The luxS-like signaling mechanism of quorum sensing also plays a role in the regulation of the cpm operon. Indeed, luxS, which is involved in the production of a newly identified autoinducer, is responsible for repression of cpm gene expression at the end of the exponential growth phase. The importance of this carbapenem production in the ecology of P. luminescens is discussed. by Streptomyces cattleya (J. S. Kahan, F. M. Kahan, R. Goegelman, S. A. Currie, M. Jackson, E. O. Stapley, T. W. Miller, A. K. Miller, D. Hendlin, S. Mochales, S. Hernandez, and H. B. Woodruff, Program Abstr. 16th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 227, 1976). To date, over 40 naturally occurring carbapenems have been identified, the majority of which originate from streptomycetes. Carbapenems are broad-spectrum agents in their activity and relatively resistant to most of the clinically encountered bacterial ␤-lactamases (4, 18, 29). The genes responsible for the synthesis of the model carbapenem molecule, 1-carbapen-2-em-3-carboxylic acid (SQ27860, Car), which contains only an unsubstituted bicyclic ring system (6, 26), have recently been identified in two gramnegative enteric bacteria, Erwinia carotovora and Serratia sp. strain ATCC 39006. These consist of a cluster of nine genes (designed car) that encode a positive regulator of carbapenem gene expression (carR), five enzymes required for the construction of the carbapenem molecule (carABCDE), two proteins responsible for a novel ␤-lactam resistance mechanism conferring carbapenem immunity in the producing host (carFG), and a protein of unknown function (carH). Genetic investigations have shown that the expression of car genes is regulated in both strains by quorum sensing. The first gene of the carbapenem cluster, carR, encodes a transcriptional activator that allows expression of the remaining downstream genes of the car cluster. In E. carotovora, CarR responds to a quorumsensing, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), signaling molecule synthesized by the product of the unlinked carI gene. This OHHL-dependent transcriptional activation allows cells to coordinate the expression of carbapenem with cell density (22). In Serratia, the autoinducer synthase SmaI, which directs the synthesis of N-butanoyl-L-homoserine lactones (BHLs) and N-hexanoyl-L-homoserine lactone (HHL), is essential for the full transcription of the carbapenem biosynthetic genes. A second regulatory gene affecting expression of
Photorhabdus luminescens, a gram-negative luminescent gamma-proteobacterium, forms an entomopathogenic symbiosis with a soil nematode of the genus Heterorhabditis. This bacterium undergoes a complex life cycle that involves a symbiotic stage, in which bacteria colonize the intestine of the nematodes, and a pathogenic stage, in which susceptible insects are killed by the combined action of the nematode and the bacteria. After entering the insect host, the nematodes expel their bacterial symbionts into the insect hemocoel. Once released, the bacteria proliferate, kill the insect within 24 h to 48 h, and enhance conditions for nematode reproduction by providing nutrients and other growth factors. Remarkably, the insect carcass does not putrefy over a period of several weeks, because P. luminescens produces several broad-spectrum antibiotics that prevent contamination of the insect cadaver by other microorganisms. These products are believed to be secreted into the insect hemolymph when the bacteria enter stationary-phase conditions. Two P. luminescens antibiotic molecules have now been identified. They consist of a red pigment with antimicrobial activity, identified as an anthraquinone derivative, and a hydroxystilbene derivative, 1,3-dihydroxy-4-isopropylstilbene, which showed strong fungicidal activity in previous studies (20, 30). Carbapenems are a class of ␤-lactam antibiotics synthesized by a biochemical route distinct from that found in the biosynthetic pathways of the sulfur-ring ␤-lactams, namely, penicillins, cephamycins, and cephalosporins (5, 43). As naturally produced metabolites, they were originally isolated in the late 1970s from Streptomyces species. The first carbapenem antibiotic discovered was thienamycin, produced in small quantities * Corresponding author. Mailing address: Unite´ de Ge´ne´tique des Ge´nomes Bacte´riens, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris cedex 15, France. Phone: 33 (0) 1 45 68 84 43. Fax: 33 (0) 1 45 68 89 48. E-mail: [email protected]
† Present address: Laboratory of Microbiology, SCK/CEN, Boeretang 200, 2400 Mol, Belgium. 3780
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TABLE 1. Bacterial strains and plasmids Strain or plasmid
E. coli S17-1 E. coli TG1 P. luminescens P. luminescens P. luminescens P. luminescens
Relevant genotype ⫺
TT01 PL2101 PL2102 PL2103
pro r n Tp Sm RP4-2-Tc::Mu::Tn7 recA thi supE44 hsd⌬5 thi-1 ⌬(lac-proAB) F⬘ [traD36 proAB⫹ laclq lacZ⌬M15] Wild type TT01 cpmA::Gm TT01 luxS::Cm TT01 slyA::Kan
E. coli BCSE 8635a Klebsiella pneumoniae 4321a Enterobacter cloacae T39180297a Enterobacter cloacae 39177636a Enterobacter cloacae C1a Enterobacter cloacae C1Ra Xanthomonas maltophiliaa Pseudomonas aeruginosa A81a Pseudomonas aeruginosa 39170143a
Resistant to ␤-lactam antibiotics Penicillinase producer Cephalosporinase producer Cephalosporinase derepressed Cephalosporinase producer Cephalosporinase derepressed Imipenemase producer Resistant to imipenem Resistant to imipenem
V. harveyi BB152 V. harveyi BB170
pBluescript SK⫹ pJQ200KS pDIA600
Apr cloning vehicle (ColE1 replicon) Gmr sacRB mob oriV (p15A replicon) 0.85-kb XbaI-PstI fragment (contains an internal region of cpmA) inserted in pJQ200KS 2.73-kb PstI-XbaI fragment (contains luxS::Cm) inserted in pJQ200KS 2.61-kb ApaI-SacI fragment (contains slyA::Kan) inserted in pJQ200KS
pDIA602 pDIA603 a
Reference or source
35 33 12 This work This work This work
2 2 Stratagene 27 This work This work This work
Clinical isolate is from I. Podglajen and E. Collatz.
carbapenem in Serratia (the rap gene) and E. carotovora (its homolog hor) has also been identified elsewhere (39, 40). In this report the characterization for P. luminescens of a cluster of genes (named cpmA to cpmH) responsible for the biosynthesis of a carbapenem-like antibiotic is described. Although this cluster shows significant sequence similarities with the car operon, previously identified in E. carotovora and Serratia sp. strain ATCC 39006, it displays unique features and appears to be regulated differently than in the latter two species. MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains used were TG1 (33) for plasmid maintenance and S17-1 (35) for conjugation. The various ␤-lactam indicator strains used in this study were kindly provided by Isabelle Podglajen and Ekkehard Collatz (INSERM E0004—Laboratoire de Recherche Mole´culaire sur les Antibiotiques, UFR Broussais-Ho ˆtel Dieu and Pitie´ Salpe´trie`re, Universite´ Paris VI, Paris, France). Vibrio harveyi strains are a generous gift from Bonnie Bassler (Department of Molecular Biology, Princeton University, Princeton, N.J.). Strains were routinely grown in Luria-Bertani (LB) medium at 37°C, except those of P. luminescens and V. harveyi, which were grown at 30°C in Schneider medium (Bio-Whittaker) and autoinducer bioassay (AB) medium (16), respectively. The final concentrations (in milligrams per liter) of antibiotics used for selection for E. coli and P. luminescens were as follows: gentamicin, 30; kanamycin, 20; and chloramphenicol, 20. All experiments were performed in accordance with the European requirements concerning the contained use of genetically modified organisms of group I (agreement no. 2736 CAII). Carbapenem plate assay. LB plates were spot inoculated with 24-h broth cultures of each strain to be tested and incubated for 72 h. Ten milliliters of sterile soft agar was inoculated with 100 l of the indicator strain and poured onto the test plates, which previously had been exposed to chloroform for 2 h to kill the test colonies. An inhibition zone around a spot indicated antibiotic production.
Assay for production of luxS-based signaling molecules. An AI-2 bioassay using V. harveyi BB170 was performed as described previously (36) with some modifications. Briefly, V. harveyi BB170 was grown overnight with aeration at 30°C in AB medium and diluted 1:2,500 in fresh AB medium, and 27 ml of the diluted culture was added to 3 ml of cell-free P. luminescens supernatants in a 250-ml Erlenmeyer flask. Cultures were shaken at 200 rpm in a rotary shaker at 30°C, and light emission by V. harveyi BB170 was measured every 30 min with a photomultiplicator tube (PMT H5783 model; Hamamatsu) (41). Cell-free culture fluids were prepared from P. luminescens parental and luxS mutant strains by centrifugation (2 min at 4,500 ⫻ g) and filtration of supernatants through Millex-GP 0.22-m-pore-size filters (Millipore). The supernatants were stored on ice before being assayed for luxS-based quorum-sensing activity. Three milliliters of Schneider sterile medium and V. harveyi BB152 cell-free culture fluid were used as negative and positive controls, respectively. DNA manipulations and plasmid construction. Chromosomal DNA preparation, ligation, E. coli electroporation, and Southern blotting were carried out by standard procedures (33). Plasmid DNA was isolated with the GenElute Plasmid Miniprep kit (Sigma). Restriction enzymes were obtained from Roche, and enzymatic reaction mixtures were purified with the MinElute Reaction Cleanup kit (Qiagen). To construct the pDIA600 plasmid, a DNA fragment containing an internal region of cpmA was generated by PCR with primers CpmA1 (5⬘-AAA CTGCAGCCTGCTCCATGAGTTGAAAA-3⬘) and CpmA2 (5⬘-GATCTAGT GTGCTTAGTGATGTGGA-3⬘) and genomic DNA from P. luminescens TT01. The resulting 900-bp amplified fragment was purified with the QIAquick PCR purification kit (Qiagen) and restricted by XbaI (located inside the amplified sequence) and PstI (present in primer CpmA2), before being cloned into the pJQ200KS vector (27) in an inverse orientation with respect to the lacZ promoter. Plasmid pDIA602 was constructed via two steps of PCR amplification. Briefly, the chloramphenicol acetyltransferase gene of pACYC184 (Biolabs) was amplified by PCR with oligonucleotides Cat5 (5⬘-TTGATCGGCACGTAAGA GGT-3⬘) and Cat3 (5⬘-AATTTCTGCCATTCATCCGC-3⬘), resulting in an 850-bp DNA fragment. Two 950-bp DNA fragments containing either the 5⬘ upstream region of luxS or the end of the coding region of luxS and the downstream region were also PCR generated with either oligonucleotides luxS1 (5⬘AAACTGCAGCTTTGGATGGGTGATCCCAT-3⬘) and luxS2 (5⬘-ACCTCTT ACGTGCCGATCAAGGATCCTCATCGTATCCTCACGTTCC-3⬘) or oligonucleotides luxS3 (5⬘-GCGGATGAATGGCAGAAATTGGATCCGGCT
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ATGGCTGATGTCTTGA-3⬘) and luxS4 (5⬘-AGCTCTAGACGGCGACATTA TGGTTCCCC-3⬘). The 21st bases of primers luxS2 and luxS3 are complementary to primers Cat5 and Cat3, respectively. After purification and quantification, 100 ng of each of the three previously amplified fragments was mixed and used as a template to generate a new 2,730-bp DNA fragment by a second PCR amplification with oligonucleotides lux1 and lux4. The latter amplicon, which corresponds to a luxS::Cm fragment, was purified, PstI and XbaI restricted, and ligated to the pJQ200KS vector (27) to yield pDIA602. To construct plasmid pDIA603, a 1.35-kb DNA fragment containing slyA was generated by PCR with primers SlyA10 (5⬘-AAACTGCAGCGTGAAGTATTGCGACTGTGC-3⬘) and SlyA8 (5⬘-CCAAAATGTTCAGGGAGCTCTTAACAC-3⬘) and genomic DNA from P. luminescens TT01. The amplified fragment was restricted by NaeI and SacI (both sites located inside the amplified sequence) and inserted at the SmaI and SacI sites of plasmid pBluescript SK⫹. A kanamycin resistance cassette obtained by BamHI restriction of pUC4K (Amersham Pharmacia Biotech) was then inserted into the latter vector at a BglII site located at the beginning of the slyA coding region. The slyA::Kan fragment was then cloned as an ApaI-SacI fragment at the same sites of the multiple cloning site of pJQ200KS to generate plasmid pDIA603. Mutant construction. P. luminescens mutant PL2101 was obtained by using vector pDIA600 delivered into strain TT01 by conjugal transfer from E. coli strain S17-1. Gentamicin-resistant colonies containing an integrated copy of this vector inside the cpmA locus were selected. Disruption of cpmA was confirmed by Southern blot hybridization with a PCR-amplified digoxigenin-labeled cpmA gene probe performed with oligonucleotides CpmA1 and CpmA2 with the PCR digoxigenin probe synthesis kit (Roche) (data not shown). P. luminescens luxS and slyA mutants, PL2102 and PL2103, respectively, were created via allelic exchange with plasmids pDIA602 and pDIA603, respectively. Both plasmids were transformed into E. coli S17-1 and introduced into P. luminescens by mating. Cmr Gms Sacr or Kanr Gms Sacr exconjugants were selected on proteose peptone agar (1% proteose peptone, 0.5% NaCl, 0.5% yeast extract, 15% agar) containing 2% sucrose. Insertions were confirmed by Southern blot analysis (data not shown). Handling of RNA. E. coli and P. luminescens total RNA was prepared from 10-ml cultures. Samples of bacterial cultures were collected by centrifugation for 2 min at 4°C at 6,000 ⫻ g and stored at ⫺80°C to prevent RNA degradation. Bacterial pellets were resuspended in 500 l of 25 mM Tris-HCl (pH 7.6)–60 mM EDTA solution and transferred into tubes containing 500 l of acid phenol (pH 4.5) and 0.4 g of 0.1-mm-diameter glass beads (Sigma). Cells were mechanically broken with a Fastprep apparatus (Bio 101) as homogenizer (two 30-s cycles of homogenization at maximum speed with 1-min intervals on ice). RNA was then extracted and purified with the TRIzol reagent kit (Gibco-BRL) according to the manufacturer’s recommendations. After isopropanol precipitation, RNA was redissolved in 100 l of TE (10 mM Tris, 1 mM EDTA, pH 7.6) and incubated with RNase-free DNase I (DNA-free kit; Ambion) according to the manufacturer’s protocol. RNA samples were then quantified by measuring A260 and A280. Both RNA purity and integrity were controlled by separating a sample on an agarose gel to ensure that mRNA, tRNA, and rRNA precursors could be seen. Primer extension. Reactions were performed according to standard procedures (33) with some modifications. Ten picomoles of end-labeled oligonucleotide was precipitated with ammonium acetate and ethanol at ⫺20°C, washed with 70% ethanol, dried, and resuspended in 40 l of diethylpyrocarbonate-treated TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6) to a concentration of 2.5 ng/ml. Ten nanograms of primer was annealed with 50 g of total RNA in avian myeloblastosis virus reverse transcriptase reaction buffer (Roche) and 1 mM deoxynucleoside triphosphates at 65°C for 10 min. The reaction was kept going while the temperature slowly decreased to 42°C. RNasin (20 U; Promega) was added, and the reaction was performed with 40 U of avian myeloblastosis virus reverse transcriptase (Roche) at 42°C for 90 min. One microliter of 0.5 M EDTA (pH 8) and 1 l of DNase-free pancreatic RNase (Roche) were added, and the reaction mixture was further incubated at 37°C for 30 min. The reaction mixture was precipitated with ammonium acetate and ethanol, washed with 70% ethanol, and resuspended in formamide loading buffer. As a reference, sequencing reactions were performed with the Thermosequenase radiolabeled terminator cycle sequencing kit (Amersham) with the same primer used in primer extension experiments. The oligonucleotide used to map the 5⬘ termini of cpm mRNA is CpmA7 (5⬘-CGGAAGAGTATGGTTCCAGTA-3⬘). Oligonucleotide labeling. The oligonucleotides used in primer extension experiments were end labeled with phage T4 polynucleotide kinase (BioLabs) and [␥-32P]ATP (3,000 Ci/mmol) according to standard procedures (33). Nucleotide sequence accession numbers. The 6,799- and 2,311-nucleotide sequences containing cpm clusters of P. luminescens have been assigned EMBLEBI nucleotide sequence database (European entry point of the International
APPL. ENVIRON. MICROBIOL. Nucleotide Sequence Database) accession no. AJ457087 (cpmABCDEFGH) and AJ457088 (cpmIJK). Nucleotide sequences of P. luminescens luxS and slyA genes have been assigned database accession no. AJ457090 and AJ457089, respectively.
RESULTS Identification of a carbapenem locus (car homologs) in P. luminescens. The genomic sequence of P. luminescens TT01 was recently completed at the Pasteur Institute (unpublished results). The availability of the sequence presented the opportunity to screen the genomic database for P. luminescens genes that exhibit some homology with known antibiotic genes. Using BLASTP as an analytical tool, we identified a cluster of eight coding sequences showing a high degree of similarity with proteins involved in carbapenem biosynthesis in E. carotovora. This similarity was especially obvious for three genes known to be essential for carbapenem biosynthesis, carABC (23). As shown in Fig. 1, the organization of the eight genes, here designated as cpmA to cpmH, is similar to the car loci found in E. carotovora and Serratia marcescens (24, 40). This specific nomenclature was used to avoid confusion with the universally known genes carA and carB encoding carbamoyl synthase subunits. The eight predicted proteins are similar in size and share significant amino acid identities with those described for E. carotovora. The organization of the cpmA to cpmH genes is strongly suggestive of an operon with several examples of likely translational coupling. A unique substantial gap (77 bp in P. luminescens, 55 bp in the other species) in the cpmA to cpmH region was observed between the third and fourth genes, cpmC and cpmD. Moreover, the last three genes, cpmFGH, have strong consensus signal sequences indicating a probable extracellular location. Finally, as in Erwinia and Serratia (24, 40), the translational initiation codon of the cpmA gene is not the more common ATG codon, but instead the TTG codon, suggesting that the synthesis of CpmA may be maintained at levels below those of the other Cpm proteins encoded by the same transcript. Interestingly, the P. luminescens cpm cluster displays two major differences from those found in Erwinia and Serratia strains. First, the screening of the genomic database revealed that P. luminescens possesses a second copy of the last three genes, cpmFGH, at a distinct locus (named cpmIJK). Both copies exhibit more than 98% identity at the DNA level. Differences are mainly found at the beginning of the cpmF gene, as the cpmI copy is slightly shorter, starting at a TTG codon rather than at the more common ATG. It should also be noted that both P. luminescens cpmH and cpmK genes are located upstream from a small coding sequence which is significantly similar to a portion of a putative transposase B (data not shown). This suggests that these loci may have been part of a mobile genetic element. Secondly, carR, the gene located upstream of the carA to carH genes in Erwinia and Serratia, was not found in P. luminescens. carR codes for a transcriptional regulator of the LuxR family that activates transcription of the car cluster in these bacteria. In E. carotovora, CarR responds to an acyl-homoserine lactone (AHL) derivative, a cell-cell signaling molecule, synthesized by the product of the unlinked carI gene, a LuxI homolog involved in quorum sensing (22).
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FIG. 1. Comparison of the putative P. luminescens cpm operon with the car operon of E. carotovora and Serratia sp. strain ATCC 39006. (A) Map of the genes involved in carbapenem biosynthesis. (B) Amino acid sequence identity between Car and Cpm proteins of E. carotovora, Serratia sp., and P. luminescens.
Functional role of P. luminescens cpm operon. On the basis of homology, we hypothesized that the cpm genes identified in P. luminescens might be involved in the biosynthesis of a carbapenem molecule as well as in carbapenem resistance. To obtain evidence about the presence of a carbapenem-like antimicrobial activity in P. luminescens, we tested diverse indicator strains for their sensitivity to P. luminescens (Table 1). These strains are clinical isolates that have developed resistance to ␤-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. An overlay of each indicator strain was poured onto an LB plate containing spotted P. luminescens colonies previously killed by exposure to chloroform. As shown in Fig. 2A, inhibition halos caused by antibiotic-induced cell killing were reproducibly observed with strains of E. coli, Klebsiella pneumoniae, and Enterobacter cloacae. No antibiosis was observed with the various imipenem-resistant clinical isolates of Pseudomonas aeruginosa, which have developed resistance to carbapenem by reducing the permeability of the outer membrane (32). Very small antibiosis zones were observed with a lawn of Xanthomonas maltophilia, a potent imipenemase strain. These results are consistent with the hypothesis that P. luminescens is a carbapenem producer and that Enterobacter cloacae is the species most sensitive to this ␤-lactam compound. Antibiosis assays performed with an isogenic set of Enterobacter cloacae strains producing low or high levels of cephalosporinase confirmed our observations (Fig. 2B). Indeed, the parental strain exhibited a growth inhibition in the presence of P. luminescens while no inhibition halo was observed with the cephalosporinase derepressed derivative. These latter strains were therefore chosen to test carbapenemlike activity in the rest of this work. To ascertain that the carbapenem-like activity observed was
related to cpm genes, a mutant of P. luminescens (PL2101) was constructed. A suicide vector in P. luminescens was designed to disrupt cpmA encoding the putative carbapenem synthetase and to simultaneously deprive the remaining part of the cpm cluster of a promoter (see details in Materials and Methods). This plasmid (pDIA600) was delivered to strain TT01 by conjugal transfer from E. coli strain S17-1. P. luminescens colonies that had integrated the vector inside the cpmA locus by a single step of homologous recombination were selected. Afterwards, the cpm mutant, designated PL2101, was compared to the wild-type strain for carbapenem production (Fig. 2). The zone of antibiosis caused by PL2101 was not as large as that caused by the wild-type strain of P. luminescens under similar conditions. This result further supports the involvement of the cpm cluster in carbapenem biosynthesis. The inhibition halos still observed were likely due to the production of other antibiotics by P. luminescens. Characterization and expression of the cpm cluster from P. luminescens. Initial attempts to detect cpm mRNAs by reverse transcription-PCR suggested a very low level of cpmA expression (data not shown). Consequently, in order to map the transcription start point of the cpmA-to-cpmH putative operon, primer extension analysis was performed with a high quantity (50 g) of RNA. P. luminescens total RNA was extracted during exponential growth phase and hybridized with primer CpmA7 specific for cpmA. The starting point is an adenosine residue located 69 bp upstream from the translation start codon of cpmA (Fig. 3B) and is preceded by ⫺35 and ⫺10 consensus sequences (TTGATA-16 bp-TACTAT ). Interestingly, the upstream region (from ⫹1 up to ⫺100) and the 5⬘ untranslated region were particularly AT rich, 88 and 85%, respectively (Fig. 3A).
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FIG. 2. Carbapenem-like activity of P. luminescens visualized by antibiosis. Three-day LB plates spotted with 5 l of both TT01 (wild type) and mutants were inoculated with 100 l of various ␤-lactam indicator strain cultures (OD600 ⫽ 0.2) mixed in soft agar. Growth inhibition around a spot indicates production of antibiotics to which the indicator strain is sensitive. All experiments were repeated at least three times. (A) Analysis of carbapenem production and the effect of a cpmA mutation (PL2101) by antibiosis of lawns of various ␤-lactam indicator strains. (B) Analysis of the effect of various mutations on the ability to produce carbapenem by antibiosis of lawns of Enterobacter cloacae strains C1 (cephalosporinase producer) and C1R (cephalosporinase derepressed). 1, cpmA mutation (PL2101); 2, luxS mutation (PL2102); 3, slyA mutation (PL2103).
Primer extension analyses were then performed to monitor cpm mRNA abundance over a complete growth cycle at 30°C in P. luminescens (Fig. 4). This analysis revealed that early growing cells (optical density at 600 nm [OD600] ⫽ 0.3)
already contained substantial amounts of cpm mRNA, which peaked at mid-exponential growth (OD600 ⫽ 1.0). The abundance of transcripts subsequently dropped to become almost undetectable in stationary phase. Moreover, no cpm
FIG. 3. (A) Nucleotide sequence of the 5⬘ region of cpmA from P. luminescens. Promoter sequences (⫺35 and ⫺10 boxes) are boxed. The Shine-Dalgarno sequence (SD) is underlined. Only the first codons of the coding sequence of cpmA are shown in boldface. (B) Primer extension analysis of cpmA transcripts in P. luminescens. Total RNA was extracted from an exponential culture of P. luminescens TT01 grown at 30°C in Schneider medium (OD600 ⫽ 1.4). The arrow indicates the position of the extension product obtained.
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FIG. 4. (A) Primer extension analysis of cpm mRNA at various times during P. luminescens growth at 30°C in Schneider medium. Lanes: 1, early log growth (OD600 ⫽ 0.3); 2, log growth (OD600 ⫽ 1.0); 3, early stationary phase (OD600 ⫽ 4.0). Equal amounts of each RNA sample (50 g) were used. Experiments were done in triplicate. The relative cpm mRNA abundances at different times during P. luminescens growth, with the lowest mRNA level taken as 1, are represented graphically. (B) P. luminescens growth curve indicating the different times when mRNAs were extracted. 1, early log growth (OD600 ⫽ 0.3); 2, log growth (OD600 ⫽ 1.0); 3, early stationary phase (OD600 ⫽ 4.0). Since P. luminescens synthesizes a variety of secondary metabolites which interfere with OD measurement at late exponential phase, correlations between CFU and OD measurements were used to define the various growth stages.
mRNA was found in 15-, 24-, or 48-h-old cultures (data not shown). A quorum sensing-dependent regulation? To extend our analysis of the cpm operon, the role of quorum sensing in the regulation of its expression was examined. In Erwinia and Serratia species, it is known that CarR, the product of the first gene of the cluster, activates transcription of the remaining genes in response to a specific AHL synthesized by unlinked genes encoding LuxI homologs (CarI and SmaI in Erwinia and Serratia, respectively) (22, 23, 40). Mutations in either gene lead to the abolition of carbapenem synthesis. In P. luminescens, no carR homolog was identified, either immediately upstream of cpmA to cpmH or elsewhere on the chromosome. In addition, the screening of our database revealed that the P. luminescens genome does not contain any protein showing homology with members of the LuxI or LuxM family of autoinducer synthases, whose function is to catalyze the formation of various AHLs involved in quorum sensing (11). These results suggest that P. luminescens does not synthesize any AHL derivative. Therefore, significant differences in the regulation of carbapenem production may exist between this bacterium and Erwinia and Serratia species.
CARBAPENEM BIOSYNTHESIS IN P. LUMINESCENS
FIG. 5. P. luminescens production of a luxS-dependent AI-2 activity. (A) AI-2 bioassay of P. luminescens culture supernatants with V. harveyi BB170 as reporter. Ten percent cell-free supernatants were mixed with the reporter strain and incubated at 30°C on a rotary shaker. Light production was determined 4 h 30 min after the BB170 cells were mixed with the various cell-free supernatants (OD600 ⫽ 0.1). Cell-free culture fluids were prepared from P. luminescens parental (TT01) and luxS mutant (PL2102) strains grown to an OD600 of 1.0. Schneider sterile medium and V. harveyi BB152 cell-free culture fluid were used as negative and positive controls, respectively. (B) Inhibition of growth by P. luminescens cell-free supernatants. Cell-free supernatants were prepared from a wild-type P. luminescens strain grown in Schneider medium for 5 h (exponential phase; OD600 ⫽ 1.0) (open circles), 10 h (early stationary phase; OD600 ⫽ 4.0) (open triangles), and 15 h (stationary phase) (open diamonds). Ten percent (vol/vol) cell-free supernatants (open symbols) or sterile Schneider medium (filled squares) was added to the V. harveyi BB170 reporter strain, and cultures were incubated at 30°C on a rotary shaker.
Although the AHL-based quorum sensing appears to be the predominant quorum signal used by host-associated gram-negative bacteria, another signaling pathway related to an alternate autoinducer (termed AI-2) has recently been discovered (34, 37). A gene involved in the new autoinducer synthesis, luxS, has been found in a large number of bacteria (19, 37). One homolog of luxS was identified in the P. luminescens genome, suggesting that P. luminescens directs the synthesis of this autoinducer. To examine whether P. luminescens produces such a molecule, we tested the ability of medium conditioned by P. luminescens cells to induce luminescence in the V. harveyi reporter strain BB170 (36). The results shown in Fig. 5A demonstrate that the addition of P. luminescens cell-free supernatant stimulates light emission in V. harveyi reporter strain BB170, while bioluminescence of BB170 was not increased by supernatant prepared from the luxS mutant PL2102 (see below). The level of luminescence induced by P. luminescens
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supernatant was about 15-fold lower than that measured upon addition of a positive control, V. harveyi BB152 cell-free supernatant. It has to be mentioned that, in contrast to V. harveyi BB152, P. luminescens supernatant was prepared from cells grown to a low cell density to preclude the production of antibiotics. Indeed, media conditioned by P. luminescens grown to higher cell density strongly inhibit V. harveyi BB170 growth (Fig. 5B). These results indicate that P. luminescens produces the new autoinducer AI-2 during exponential growth phase and that luxS is involved in this production. This prompted us to evaluate the influence of the new pheromone-mediated pathway on carbapenem synthesis. For this purpose, a chromosomal null luxS mutant (PL2102) was constructed via allelic exchange with pDIA602, a suicide plasmid that harbors a chloramphenicol acetyltransferase cassette flanked by the 5⬘ and 3⬘ regions of the luxS gene (see Materials and Methods). The luxS mutation did not affect P. luminescens growth rate in Schneider medium, although the mutant growth resumed with a short delay after inoculation into a fresh medium (Fig. 6A). The propensity of P. luminescens to produce pigments was also affected (data not shown). The mutant (PL2102) was compared to the wild-type strain for its carbapenem-like activity (Fig. 2B). Under similar conditions, the antibiosis zone produced by the luxS mutant was reproducibly larger than that of the wild-type strain, suggesting that PL2102 produces more antibiotic. Data from three independent primer extension analyses revealed that luxS exerts a repressor effect on carbapenem biosynthesis gene expression at the end of the exponential growth phase (Fig. 6B). In contrast to the wildtype strain, where the abundance of the cpm transcript rapidly declined (up to 40-fold) before entry into stationary phase (OD600 ⫽ 4), the cpm mRNA level in PL2102, although decreasing after mid-exponential growth (up to fivefold), remained higher for a quite longer period (Fig. 6B), being still faintly detectable after 20 h of growth (data not shown). SlyA, a Rap/Hor homolog, also controls carbapenem production in P. luminescens. In E. carotovora and S. marcescens, carbapenem production is also regulated by another regulatory protein, designed Rap in Serratia spp. and Hor in Erwinia spp. (39). Mutations in the corresponding genes resulted in strains defective in carbapenem production. We identified such a regulator in P. luminescens and named it SlyA, according to its high amino acid identity with the E. coli and Salmonella enterica serovar Typhimurium Rap/Hor homolog, SlyA (for salmolysin) (21). To test its putative regulatory function on cpmA to cpmH expression, a slyA::Kan mutant (PL2103) was constructed by allelic exchange with pDIA603, a plasmid that harbors a copy of the slyA gene disrupted at the beginning of the coding region by a kanamycin cassette (see Materials and Methods). The mutant (PL2103), which showed a growth rate similar to that of the wild-type strain (Fig. 6A), was next compared to this strain for its carbapenem-like activity (Fig. 2B). The zone of antibiosis caused by the mutant on an Enterobacter cloacae lawn was not as large as that caused by the wild-type strain, suggesting that PL2103 produces less antibiotic than does the wild-type strain. To confirm this observation, primer extension analysis was performed on total RNA extracted from exponential-growth-phase culture of the wild-type and PL2103 strains (Fig. 6C). The amount of cpm mRNA measured in
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FIG. 6. Analysis of the effect of luxS and slyA mutations on growth rate and cpm mRNA synthesis. (A) Growth curve comparison among TT01 (filled squares), PL2102 (luxS mutant) (open triangles), and PL2103 (slyA mutant) (open circles) strains of P. luminescens. (B) Primer extension analysis of the effect of luxS mutation on cpm mRNA synthesis. RNA samples were taken at different times from cultures of TT01 and PL2102. Lanes: 1, log growth (OD600 ⫽ 1.0); 2, early stationary phase (OD600 ⫽ 4.0). (C) Primer extension analysis of the effect of slyA mutation on cpm mRNA synthesis. RNA samples were taken from exponential-growthphase cultures of TT01 and PL2103 (OD600 ⫽ 0.9). Three independent primer extension analyses were performed. Equal amounts of each RNA sample (50 g) were used. The relative cpm mRNA abundances measured in TT01 and mutants, with the lowest mRNA level taken as 1, are represented graphically. Error bars represent standard errors of the mean. Statistical analysis was performed with a Student t test, resulting in a significant difference (P ⬍ 0.05).
PL2103 was less (about twice) than that in the untransformed strain. SlyA seems, therefore, to positively control the production of a carbapenem-like molecule in P. luminescens, like Hor and Rap in E. carotovora and S. marcescens, respectively.
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DISCUSSION The rapid emergence of antibiotic-resistant bacterial infections underscores a growing need for novel effective therapeutic agents. Although antibiotic production is known to be a very common characteristic of bacteria associated with insectpathogenic nematodes such as Photorhabdus species, no systematic chemical study of these antibiotics has yet been undertaken, nor are the genes involved in antibiotic biosynthesis known in these species. We screened the recently completed P. luminescens genomic database to identify loci showing sequence homology to genes known to be involved in antibiotic biosynthesis. By using BLASTP comparison, eight genes (cpmA to cpmH) putatively responsible for carbapenem production were identified. Antibiotic plate assays with various ␤-lactam-resistant indicator strains confirmed the production of a carbapenem-like molecule by P. luminescens. This compound was shown to be effective against E. coli, K. pneumoniae, and Enterobacter cloacae. It was ineffective against imipenem-resistant clinical isolates of Pseudomonas species and had little, if any, inhibitory effect on X. maltophilia. Our results also showed that carbapenem is clearly not the sole antibiotic produced by P. luminescens. Indeed, growth inhibition zones were still observed in the presence of a cpm mutation. Furthermore, the carbapenem-like molecule produced by P. luminescens is not effective against gram-positive bacteria, as no difference in growth inhibition halo size could be detected in antibiosis assays performed with bacteria of this class and either a wild-type or a cpm mutant strain of P. luminescens. The two strains similarly inhibit gram-positive bacterial growth (data not shown). The discovery of a new ␤-lactam antibiotic synthesized by the TT01 strain enlarges the spectrum of antibiotics known to be produced by P. luminescens. It is thought that the role of antibiotic production by P. luminescens is in keeping the insect carcass from putrefying after the bacterium-nematode infection, thereby maintaining a pure culture of P. luminescens inside the infected insect. This is a necessity for the successful completion of the nematode life cycle and for symbiont transmission to the nematode’s progeny. Indeed, in the insect hemolymph, the infective nematode develops into hermaphrodites with eggs, but its offspring is unable to develop beyond the first juvenile stage (J1) without the presence of the symbiotic bacteria (17). When the symbiont dominates the microbial flora, nematodes subsequently develop into the nonfeeding infective juvenile stage (DJ), retain the symbiotic bacteria in the intestine, and emerge from the insect carcass in search of a new host (13). Comparative sequence analysis of the cpm cluster of P. luminescens with those found in E. carotovora and Serratia spp. revealed unique features. The P. luminescens genome contains two copies of the last three genes, i.e., cpmFGH and cpmIJK, at distinct loci. The cpmIJK cluster is located near a gene homologous to penicillin binding protein B (38) and several genes showing homology to bacteriocin and immunity proteins (31). This location suggests that the yet-uncharacterized carbapenem self-resistance mechanism encoded by cpmF and cpmG may have a more general role. Although there is no evidence for the involvement of cpmH in either carbapenem biosynthesis or carbapenem resistance (23), its position immediately downstream of cpmF and cpmG, even at the second cpm un-
related locus, strongly suggests that it is involved in some aspect of carbapenem resistance. Remarkably, carR, the gene located upstream of the carA to carH genes in Erwinia and Serratia, was not found in P. luminescens. The absence of this AHL-dependent activator suggests that the mechanism by which P. luminescens regulates the biosynthesis of its carbapenem-like compound is different from that described for Erwinia and Serratia. Carbapenem synthesis is not induced by AHL-based quorum sensing in P. luminescens. The finding that the P. luminescens genome does not contain any luxI homolog, the gene responsible for AHL synthesis, is surprising. Indeed, AHL appears to be the predominant quorum signal used by host-associated gram-negative bacteria (11), and it has been detected in Xenorhabdus nematophilus (10), another entomopathogenic bacterium symbiotically associated with nematodes (13). Moreover, as P. luminescens is a luminescent bacterium, it is expected that lux gene expression is controlled by the production of this autoinducer as in marine bacteria (9, 14). However, in accordance with the result of our BLAST comparisons, previous experiments looking for the accumulation of inducing activity have shown no indication of control of luminescence via autoinduction by AHLs in P. luminescens (14). As with the Erwinia and Serratia car genes, the cpmA to cpmH expression in P. luminescens is positively regulated by a Rap/Hor homolog, SlyA (40). However, rather than a defective phenotype observed in those species, slyA disruption resulted only in a lower carbapenem production in P. luminescens. SlyA is a member of a growing family of transcriptional regulators which control diverse physiological processes in human, animal, and plant pathogens (28, 39). The mechanism by which they exert their influence is currently unknown (8, 42), as members of this family do not have any of the motifs common among other regulatory proteins such as helix-turn-helix motifs or zinc finger proteins (25). Further studies are therefore necessary to understand the nature of SlyA influence on carbapenem biosynthesis genes in P. luminescens. Finally, the luxS-based quorum-sensing system, used by a variety of bacteria for communication (37), was shown to control the production of a carbapenem-like compound in P. luminescens. The luxS-mediated pathway negatively controls carbapenem biosynthesis gene expression. The cpmA mRNA amount detected in PL2102 was higher than that found in the wild type, especially during late-exponential growth phase. This quorum-sensing regulatory mechanism likely functions to limit carbapenem biosynthesis gene expression to the exponential growth phase. This timing must be important to ensure the monoxenic conditions for nematode reproduction at the moment of the insect death (about 24 to 48 h after infection). At this stage, P. luminescens bacteria have been growing rapidly and progressively enter into stationary phase (7). Carbapenem production before this period likely allows P. luminescens to inhibit most of the insect gut microflora and to avoid therefore their invasion of the insect body at the moment of insect death. Carbapenem secreted by P. luminescens is indeed active mainly against gram-negative bacteria and especially the family Enterobacteriaceae. It could also itself be used as some type of quorum sensing, when the bacterial concentration is still low. Furthermore, it may be important for bacteria not to be intoxicated by their own antibiotic production, since they are con-
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fined to a close medium. Other antibiotics produced may help to control the microflora or, if they are produced later, may be important to prevent contamination of the cadaver by other soil or water microorganisms. In contrast with P. luminescens, where carbapenem production occurs in exponential growth phase, cell-density-dependent regulation in Erwinia and Serratia allows carbapenem to be maximally expressed only at relatively high cell concentrations (1, 40). In Erwinia, harnessing the regulation of virulence factors and carbapenem to population size ensures that plant defenses are quickly overwhelmed while minimizing bacterial competition for ensuing resources by antibiotic production. P. luminescens, unlike Erwinia, does not have to quickly overwhelm host defense, as it uses a protective vector, its nematode, to be inoculated directly into the insect open blood circulation system. Moreover, its vector, which is not recognized as nonself by the host immune system, possesses several weapons (i.e., helminthic toxins and an immune-depressive factor) against the insect defense system to protect its bacterial symbiont (3, 15). The complete elucidation of the genome sequence of strain TT01 of P. luminescens would allow us to identify other loci involved in the biosynthesis of the P. luminescens antimicrobial agents active against gram-positive bacteria, yeasts, and some fungi, as well as genes encoding potential antivertebrate virulence factors or a specific mechanism for self-protection. This will undoubtedly contribute to a better understanding of the biology of this insect pathogen.
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13. 14. 15. 16. 17. 18. 19. 20. 21.
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