1 Unité de Génétique des Génomes Bactériens, Département de Structure et Dynamique des Génomes, Institut Pasteur, 75724 Paris Cedex 15, France
2 Laboratoire de Génomique des Micro-organismes Pathogènes, Département de Structure et Dynamique des Génomes, Institut Pasteur, 75724 Paris Cedex 15, France
3 Plate-forme Technologique Protéomique, Institut Pasteur, 75724 Paris Cedex 15, France
Correspondence
Sylviane Derzelle
sderzell{at}jouy.inra.fr
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ABSTRACT |
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The GenBank/EMBL accession number for the astRS sequence reported in this paper is AJ510203.
Present address: INRA, Unité de Biochimie et Structure des Protéines, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.
Present address: Laboratoire de Dynamique, Évolution et Expression de Génomes de Micro-organismes, FRE 2326, Université Louis Pasteur/CNRS, 67083 Strasbourg Cedex, France.
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INTRODUCTION |
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Photorhabdus spp. have the somewhat unusual ability to exist in two phenotypically distinct forms, known as primary variant (form I) and secondary variant (form II) (Akhurst, 1980; Boemare & Akhurst, 1988
; Boemare et al., 1997
). The two variants are equally pathogenic for the insects, but differ in a wide range of characteristics, including their biochemical properties and their colonial and cellular morphologies. Variant I is characterized by the production of antimicrobial agents, lipases, phospholipases, proteases, pigmentation and bioluminescence. It has distinct colony morphology, adsorbs certain dyes and develops large intracellular protein crystal inclusions (Forst et al., 1997
). Most of these characteristics specific to the primary form are essential for the symbiotic interaction between the nematode and the bacterium (Joyce & Clarke, 2003
). As expected, variant I is the bacterium normally found in association with the symbiotic infective-phase nematodes. The secondary variant either lacks or has reduced levels of the previously listed properties, and secondary variants from some strains are unable to support nematode growth and reproduction in the insect cadaver (Ehlers et al., 1990
). Upon entering the stationary phase, the secondary form also differs in its assimilation of nutrients and its vitamin requirements. Variant II cells maintain considerably higher levels of the major respiratory enzymes than do their variant I counterparts and have considerably higher levels of transmembrane proton-motive force (Boemare et al., 1997
; Smigielski et al., 1994
). As they display higher levels of cellular metabolism and respiration, variant II cells grow faster than variant I cells. Furthermore, following periods of starvation, variants II resume growth within 24 h of the addition of nutrients, compared to 14 h for variant I cells (Bleakley & Nealson, 1988
; Boemare et al., 1997
). Variant II appears spontaneously at high frequency during the stationary period of in vitro culture or during nematode rearing on an artificial diet. Although the secondary form has also been observed in vivo during the reproduction in insects (Hurlbert, 1994
), it is counter-selected by the infective juveniles which do not retain variant II bacteria in their intestinal tract. Stress conditions such as prolonged culture time, low oxygen levels, or low osmolarity in the liquid medium induce the formation of secondary cells (Boemare et al., 1997
; Krasomil-Osterfeld, 1995
). Based on differences in levels of respiratory enzymes and lag times, it has been hypothesized that secondary cells might be better adapted for survival as free-living organisms in the soil (Boemare et al., 1997
) and that the secondary phenotype is a response by the bacterium to environmental conditions not favouring nematode association (ffrench-Constant et al., 2003
).
Phenotypic variation in Photorhabdus is not a classical phase variation. The switch between the two forms appears to be unidirectional: only the transition from primary to secondary variant cells has been documented. Recent data indicate that several genetic loci affect phenotypic variation in different ways. Inactivation of either cipA or cipB genes, encoding the crystal proteins of Photorhabdus strain NC1, creates a variant cell type resembling the secondary variant in many respects (Bintrim & Ensign, 1998). Expression from a multicopy plasmid of ner, a gene which encodes a putative DNA-binding protein, switches the phenotype of the primary variant to that of the secondary (O'Neill et al., 2002
). Lastly, inactivation in secondary cells of a homologue of hexA restores the production of the primary-specific phenotypes, suggesting that secondary cells produce a repressor protein down-regulating the expression of primary-specific phenotypes (Joyce & Clarke, 2003
). These observations suggest the existence of a complex regulatory cascade of interacting genes controlling phenotypic variation in P. luminescens.
Entomopathogenic bacteria colonize diverse environments, including the nematode gut and the insect haemocoel, which have different physical and chemical properties. To establish culture in these locations, P. luminescens, like other bacterial pathogens (Hentschel et al., 2000), has evolved two-component signal transduction systems to induce the expression of the sets of genes needed. These systems comprise a membrane-associated sensor kinase and a cytoplasmic transcriptional regulator. In response to an external stimulus, the sensor component is autophosphorylated at a conserved histidine residue in an ATP-dependent reaction. The phosphoryl group is then transferred to the regulator component, promoting its binding to DNA (Stock et al., 2000
). This study reports the identification of a new two-component signal transduction system, AstRS, which is involved in adaptation of P. luminescens cells to the stationary phase and which affects the phenotypic variation process. Mutation of the regulator component AstR reduces the competitive advantage of variant I cells during stationary-phase conditions and systematically induces an earlier transition to the secondary phenotype. We hypothesize that this system may be part of the regulatory cascade tightly controlling the decision to shift to the secondary form of P. luminescens.
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METHODS |
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pDIA606 was constructed via a two-step PCR method. Briefly, the kanamycin-resistance gene of pUC4K (Amersham Pharmacia Biotech) was amplified by PCR with oligonucleotides kan5 (5'-AATTTCTGCCATTCATCCGCCACGTTGTGTCTCAAAATCTC-3') and kan3 (5'-TTGATCGGCACGTAAGAGGTATCCAGCCAGAAAGTGAGGGAGC-3'), resulting in a 1250 bp DNA fragment. Two 1075 bp DNA fragments containing either the 5' upstream region of astR or the end of the coding region of astR and the downstream region were also amplified using genomic DNA from P. luminescens TT01 and either oligonucleotides AstA3 (5'-AAACTGCAGGATGCTGGTAGCAACAGCGC-3') and AstA4 (5'-ACCTCTTACGTGCCGATCAAGGCAATGTTGCCAGTATCGTA-3'), or AstA5 (5'-GCGGATGAATGGCAGAAATTGGACTAAGCAGACGATGAGCG-3') and AstA6 (5'-CGGGATCCGCGATTCAGTGCCACATTGA-3'). The first 20 bases of primers AstA4 and AstA5 are complementary to primers kan5 and kan3, respectively. After purification and quantification, 100 ng samples of each of the three previously amplified fragments were mixed and used as template to generate a new 3360 bp DNA fragment using oligonucleotides AstA3 and AstA6. The latter amplicon, which corresponds to an astR : : kan fragment, was purified, cut with PstI and XbaI and ligated to the pJQ200KS vector (Quandt & Hynes, 1993) to yield pDIA606. To construct pDIA608, a DNA fragment containing the astR gene was generated by PCR with primers AstS3 (5'-AAACTGCAGCTGGCAAGGTATCGCACTGC-3') and AstA6, and genomic DNA from P. luminescens TT01. The resulting 1700 bp fragment was purified with the QIAquick PCR purification kit (Qiagen), cut with PstI (present in primer AstS3) and EcoRI (located inside the amplified sequence), and cloned into the pSU18 vector (Bartolomé et al., 1991
) in the same orientation as the lacZ promoter.
DNA manipulations.
Chromosomal DNA preparations, ligations, electrophoresis and Southern blotting were carried out according to standard procedures (Sambrook et al., 1989). Plasmid DNA was isolated with the GenElute Plasmid Miniprep kit (Sigma). Restriction enzymes were obtained from Roche and enzymic reactions were purified with the MinElute Reaction Cleanup kit (Qiagen). pDIA606 was introduced into P. luminescens by conjugal transfer, whereas pDIA608 was electroporated. E. coli and P. luminescens were transformed by electroporation using a Bio-Rad gene pulser according to standard procedures (Sambrook et al., 1989
).
Mutant strain construction.
Strain PL2106 was created via allelic exchange with pDIA606 (which contains a kanamycin-resistance cassette in the astR coding region). pDIA606 was transformed into E. coli S17-1 and introduced into P. luminescens by mating. CmRGmSSacR exconjugants were selected on proteose peptone agar [1 % (w/v) proteose peptone, 0·5 % (w/v) NaCl, 0·5 % (w/v) yeast extract, 1·5 % (w/v) agar] containing 2 % (w/v) sucrose. These exconjugants had undergone allelic exchange and lost the wild-type copy of astR and the plasmid vehicle. Insertions were confirmed by Southern blot hybridization using a PCR-amplified digoxigenin (DIG)-labelled astR gene probe obtained by PCR with oligonucleotides AstA5 and AstA6 and the PCR DIG probe Synthesis kit (Roche).
Handling of RNA.
Total RNA was prepared from 10 ml cultures of E. coli and P. luminescens as previously described (Derzelle et al., 2002). Primer extension reactions were performed by standard procedures (Sambrook et al., 1989
) with some modifications as previously described (Derzelle et al., 2002
). Ten nanograms of end-labelled primer was annealed with 50 µg total RNA and reverse transcription was performed with AMV reverse transcriptase (Roche) at 42 °C for 90 min. As a reference, sequencing reactions were performed with the Thermosequenase radiolabelled terminator cycle sequencing kit (Amersham) with the same primer as used to map the 5' termini of astR mRNA, AstA4. Quantification of band intensities was performed using the PDQUEST software package (PDI, Humington Station) as follows. A large and identical area including the band to be quantified was delimited for each primer extension line. The background noise of the gel was subtracted and band intensity values were normalized: the raw quantity of each band was divided by the total intensity value of all the pixels measured in the area.
Swarming capacity.
Tryptone swarm plates containing 1 % (w/v) Bacto-Tryptone, 0·5 % (w/v) NaCl and 0·3 % (w/v) Bacto-Agar were used to test bacterial motility as previously described (Bertin et al., 1999). Cell concentration of each culture was measured and adjusted to OD600 2 before 5 µl of the culture was applied to the swim plate.
Antibiotic plate assay.
LB plates were spot inoculated with 24 h-old-broth cultures of each strain to be tested and incubated for 3 days. Ten millilitres of sterile soft agar was allowed to cool to 45 °C before being inoculated with 100 µl indicator strain culture (OD600 0·2). After mixing, it was poured onto the plates, which had just been exposed to chloroform for 2 h to kill the spotted colonies. An inhibition zone around a spot indicated the production of antibiotics.
Bacterial survival assays.
Schneider medium (10 ml in a 250 ml glass Erlenmeyer flask) was inoculated with 0·3 ml of an overnight culture of P. luminescens strain TT01 grown in LB medium at 30 °C and 140 r.p.m. and incubated under the same conditions. Samples were removed periodically, diluted in Schneider medium and plated on nutrient agar plates, which were then kept in the dark at 30 °C. Starvation survival was measured by taking samples every 48 h and determining c.f.u. ml1.
In vivo pathogenicity assays.
The pathogenicity assays were performed on the common cutworm Spodoptera littoralis as previously described (Givaudan & Lanois, 2000). Briefly, a 20 µl sample of exponentially growing bacteria was diluted (104) in PBS and injected into the haemolymph of 20 fifth-instar larvae of S. littoralis reared on an artificial diet. The larvae were then individually incubated at 23 °C for up to 96 h, and the number of c.f.u. determined by plating dilutions on LB agar. About 10005000 bacterial cells were injected into insects. Insect death was monitored every 5 h.
Analytical two-dimensional (2D) gel electrophoresis.
Exponential- and stationary-phase cells (50 ml) were harvested by centrifugation. The cell pellets were washed with ED minimal medium (120 mM potassium phosphate buffer, 3 mM trisodium citrate) and resuspended in 1 ml distilled water. After DNase and RNase treatment, cells were disrupted with an FP120 FastPrep Cell disruptor (Bio 101) (twice for 30 s at maximum speed with 1 min intervals on ice). Cell debris was removed by ultracentrifugation for 60 min at 90 000 g.
Isoelectric focusing (IEF) was done with the horizontal Multiphor II system (Pharmacia) at 20 °C (Gorg et al., 1987, 1988
). For analytical gels, 60 µg protein was solubilized in 400 µl rehydration solution [0·5 % (v/v) Pharmalyte 310, 8 M urea, 65 mM DTT, 2 % (v/v) Nonidet P40], and loaded onto an 18 cm pH 47 L immobilized pH gradient strip (IPG) using the in-gel rehydration technique (Rabilloud et al., 1994
). For preparative gels, 120 µg protein was solubilized as mentioned above. For both analytical and preparative gels, focusing was performed for 3 h at 300 V, 1 h at 750 V, 30 min at 1500 V, 16 h at 2500 V and 2 h at 3500 V (total=50 kVh). The IPGs were equilibrated as previously described (Gorg et al., 1987
). The second dimension was performed with 11·5 % (w/v) SDS-polyacrylamide gels using the Protean II xi 2D Multicell system (Bio-Rad). Proteins were stained with silver nitrate and gels were digitized using a JX-330 scanner (Sharp). Digitized 2D gel patterns were edited and matched using the PDQUEST software package.
To account for non-specific variations, a minimum of six gels were run for both strains and conditions (exponential or stationary phase) using two independent protein preparations extracted from two independent cultures. Protein levels were expressed as percentage volume, which corresponds to the percentage ratio between the volume of a single spot and the total volume of all spots present in a gel. The mean intensity values of each spot were calculated on at least three gels. Spots showing large variation between replicates were not considered.
MALDI-TOF mass spectrometry and database searching.
MS analyses were performed using a matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) Voyager-DE-STR mass spectrometer (Applied Biosystems), operated in positive ion reflector mode. Protein spots of interest were cut out and digested with trypsin (Roche) as described previously (Shevchenko et al., 1996). Peptide mixtures were desalted with ZipTip C18 (Millipore) and analysed using a saturated solution of
-cyano-4-hydroxycinnamic acid (Sigma) in acetonitrile containing 1 % trifluoroacetic acid (Sigma) (50/50, v/v). The trypsin autolysis peptides were used as internal calibrants. Peptides were selected in the mass range 8003000 Da. A local copy of the MS-FIT program, developed by the University of California at San Francisco, was used to search the P. luminescens database (Duchaud et al., 2003
). Search parameters were as follows: monoisotopic masses, maximum allowed peptide mass error of 50 p.p.m., consideration of one incomplete cleavage per peptide, and oxidation of methionine. No restrictions on molecular mass or pI were made. A minimum of four matching peptides was required for protein identification in the database. To identify low-molecular-mass proteins, post-source decay (PSD) experiments were performed with the MALDI instrument.
The amino acid sequence similarity search was carried out using the BLASTP software (Altschul et al., 1990; Altschul & Lipman, 1990
).
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RESULTS |
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Characterization and expression of the astRS operon from P. luminescens
astS starts three bases downstream of the coding sequence of astR, suggesting that these two genes form a single transcriptional unit. To map the transcription start site of this putative operon, primer extension analysis was performed with 40 µg RNA. Total RNA was extracted from P. luminescens during the exponential growth phase and hybridized with primer BvgA4, which is specific to astR. The start point is located at a guanosine residue located 72 bp upstream of the translation start codon of astR (Fig. 2a) and is preceded by very poor 35 and 10 consensus sequences (TCGCTT-17 bp-GAAAAT).
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Construction and phenotypic characterization of an astR mutant
To obtain information about the functional role of the AstRS two-component system in P. luminescens, the regulator component AstR was inactivated by allelic exchange. A mutant strain, PL2106, was constructed with a plasmid harbouring a kanamycin-resistance cassette inserted into astR (see Methods). In this construct, the insert is unlikely to exert a polar effect on astS because of its convergent transcription. The astR mutation had no effect on exponential growth rate or on cell morphology. Exponential-phase cells were mainly rod-shaped, although they became increasingly pleiomorphic with the appearance of coccoid bodies during the stationary phase. Stationary-phase cells harboured one or two crystal inclusions (visualized by phase-contrast microscopy).
We then examined several phenotypic traits of PL2106. In our assays, both PL2106 and TT01 adsorbed the blue dye when incubated on NBTA (nutrient agar supplemented with 25 mg bromothymol blue l1 and 40 mg triphenyltetrazolium chloride l1), were bioluminescent and pigmented, although pigmentation was less intense in the mutant. We also compared antibiotic production by the mutant and wild-type (Fig. 3a) using various clinical bacterial isolates as indicator strains (Derzelle et al., 2002
). In similar conditions, the inhibition haloes caused by PL2106 on each indicator strain tested were smaller than those produced by the wild-type strain, demonstrating that the mutant produces less antibiotics than does TT01.
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The astR mutation induces early phenotypic variation in stationary-phase culture
When analysing PL2106, we noticed that some of the spotted colonies grown on solid medium displayed at their periphery a different morphology and pigmentation after several days of incubation at 30 °C (Fig. 3b), suggesting that part of the population had undergone phenotypic variation. Spontaneous switching is known to occur with the wild-type strain after long-term growth in artificial media (Boemare et al., 1997
). Analysis of the cells found on the periphery demonstrated that these cells were true secondary variant cells, as they were altered in all the primary-specific properties examined. They did not produce any crystal inclusions, and were impaired in bioluminescence and antibiotic production. They exhibited no evidence of adsorption of neutral red from MacConkey agar and were red when grown on NBTA (Boemare et al., 1997
). The colonies also lost the stickiness' of variant I cell colonies and were translucent with irregular edges (Forst & Clarke, 2002
), and the cells resumed growth faster than did variant I cells following periods of starvation (Bleakley & Nealson, 1988
; Boemare et al., 1997
). Moreover, the cells displayed a haemolytic activity at least twice that of the wild-type variant I, as recently described for variant II of the same strain TT01 (Brillard et al., 2002
). Finally, their phenotype was stable and they were never found to revert to variant I.
This observation prompted us to examine the survival of P. luminescens in starvation conditions in liquid medium. For this purpose, we monitored the survival of the astR mutant and the wild-type during long-term culture in Schneider medium (Fig. 5a). Every 2 days, samples of the stationary-phase cultures were diluted, plated onto nutrient agar and c.f.u. counted 72 h later. The number of variant II colonies was simultaneously counted (Fig. 5b
). The number of c.f.u. decreased steadily over 1 week for both strains, although initially the viability of the astR mutant decreased more rapidly. A plateau was then reached and about 35 % of both wild-type and astR populations were able to survive for longer periods. At the end of the first week, the number of c.f.u. in the mutant culture started to increase for a few days (Fig. 5a
); at this time, all mutant c.f.u. were composed of variant II cells (Fig. 5b
). As previously described, the colonies were secondary-like in all phenotypes tested and remained stable when subcultured. The growth observed is therefore probably due to the ability of the secondary variants to grow further in media that have been partially depleted by primary variants, as reported by Boemare et al. (1997)
. As illustrated in Fig. 5(b)
, the astR mutation induced a phenotypic shift to secondary variant of all cells in the population a few days after the culture entered stationary phase. Similar phenotypic switching did not occur in the wild-type population until the culture had been incubated at 30 °C for at least 10 days. Complementation of PL2106 with pDIA608, an intermediate copy number plasmid carrying astR, restored a wild-type behaviour (Fig. 5
). Phenotypic switching occurred at the same time in the complemented mutant culture and in the wild-type strain culture, i.e. after at least 10 days in stationary phase. The effect on phenotypic variation is therefore specific to astR.
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Comparative analysis of the 2D protein pattern associated with the astR mutation
To obtain insight into the role that AstRS plays in adaptation and survival in stationary phase, and phenotypic variation processes, we next attempted to identify the proteins that are produced under the control of the AstRS system. For this purpose, proteome modifications generated by the disruption of astR were explored using 2D electrophoresis to visualize and to identify the AstR targets. Strains PL2106 and TT01 were grown in Schneider medium to exponential or stationary phase. After disrupting the cells, proteins were separated on a 2D SDS-PAGE gel, silver stained, scanned and analysed. Representative patterns of silver-stained proteins are shown in Fig. 6. The overall profile of total soluble proteins in both strains was found to be identical during the exponential growth (Fig. 6a, b
), whereas some differences were observed in the stationary phase (Fig. 6c, d
). Several polypeptides were affected (by at least a factor of two) when astR was inactivated. Some were up-regulated, others were down-regulated. Using the whole-genome sequence data (Duchaud et al., 2003
) and MALDI-TOF MS (Shevchenko et al., 1996
), we identified these spots without ambiguity (Table 1
).
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The amounts of ten proteins decreased: (iii) two proteins similar to several universal stress proteins and highly similar to the nucleotide-binding proteins of the uspA family (we have accordingly named the two proteins UspB and UspC); (iii) UspA, the universal stress protein A, a general responder to growth inhibitory conditions, which accumulates following a large number of different environmental insults, including depletion of essential nutrients (Nyström & Neidhardt, 1994); (iv) a protein weakly similar to a hypothetical protein from Streptococcus pneumoniae (27 % identity and 50 % conservative replacement); (v) ArgB, an acetylglutamate kinase involved in the biosynthesis of arginine; (vi) the quinone oxidoreductase Qor, a protein similar to the
-crystalline protein found in the liver and kidneys of higher organisms, where it acts through a one-electron transfer process to produce the semiquinone radical (Thorn et al., 1995
); (vii) two dehydrogenases (DHs) found in the same spot LpdA, the dihydrolipoamide DH E3 (LPD) component of both pyruvate and 2-oxoglutarate DH complexes, and an aldehyde DH with some homology with putative betaine-aldehyde DH; (viii) a protein showing in its internal region some similarity to triacylglycerol lipase protein of Arabidopsis thaliana (40 % identity) and Candida ernobii (33 %); (ix) five spots identified as isoforms of a predicted aldehyde DH showing the highest amino acid identities with aldehyde DH B of Fusobacterium nucleatum (54 %); and (x) four spots identified as isoforms of the chaperonin GroEL.
In conclusion, the proteomic analysis indicated that the astR deletion affected the synthesis in stationary growth phase of proteins involved in electron-transport systems, energy metabolism and iron acquisition, as well as universal stress proteins, antioxidant protein and molecular chaperones.
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DISCUSSION |
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Nevertheless, some functional similarities exist between the two signal transduction systems. Both systems control swarming in their respective species (Akerley & Miller, 1993; Han et al., 1999
). A prominent feature of the Bvg phase is the motility phenotype. In Bordetella spp., the flagellar genes, which are regulated as a cascade at the top of which is an analogue of flhDC, are negatively regulated by BvgAS. Similarly, in P. luminescens, the astR mutant PL2106 is more motile than its parental strain. Primer extension analysis of flhDC mRNA abundance indicates that this operon is negatively regulated by AstRS in P. luminescens. In addition to flagella, both systems also control genes involved in iron acquisition. In B. pertussis, BvgA activates a TonB-dependent siderophore receptor named BfrD (Antoine et al., 2000
), whereas in B. bronchiseptica strains, the production of the siderophore alcaligin is induced in the Bvg phase (Giardina et al., 1995
). In P. luminescens, we identified two putative iron-transport proteins among the proteins up-regulated in the astR background: a probable iron(III) ABC transporter and the acidic isoform of a probable ferrichrome ABC transporter, which is likely to be involved in the acquisition of exogenous iron, such as ferrichrome.
Analysis of the AstRS system suggests that AstRS plays a role in stationary-phase adaptation and starvation survival in P. luminescens. astRS is maximally expressed at the onset of the stationary phase and 2D PAGE analysis demonstrated that the protein pattern is specifically affected. It is however unknown whether AstR, directly or indirectly, regulates the genes encoding the proteins whose abundance is altered by its inactivation. Interestingly, three universal stress proteins are down-regulated in the astR mutant background during this period: UspA and its paralogues UspB and UspC. Usp proteins have a general protective function in growth-arrested cells and are required for the management of DNA damage in cells entering stationary phase. They become some of the most abundant proteins in stationary phase (Gustavsson et al., 2002). In the astR mutant, Usp proteins are no longer induced in the stationary phase. This lack of induction may account for the more rapid decrease in survival observed for the mutant during long-term cultures. The E. coli UspA mutant is impaired in its ability to survive growth arrest.
In addition, some of the secondary metabolism molecules synthesized by P. luminescens upon entry into stationary phase, antibiotics, pigments and exoenzymes such as the putative lipase identified in 2D electrophoresis experiments, are produced in smaller amounts in PL2106. This finding is not surprising if AstRS plays a role in the adaptation of P. luminescens to stationary-phase growth. Interestingly, this decrease might be connected to the parallel decrease in several isoforms of the chaperonin GroEL in PL2106. GroEL is responsible for the folding, repair and degradation of proteins, particularly when export and translocation processes are altered (Grallert & Buchner, 2001). Some of the pleiotropic effects observed in the mutant may result from the partial loss of GroEL chaperone activities. Once again this is consistent with the nematode biotope of the bacteria. The above-mentioned molecules are surface associated or secreted; therefore they might be affected by lack of GroEL. They are not the only ones, as some universal stress proteins easily acquire unstable conformations and are very sensitive to proteolysis. In E. coli, one of these proteins (termed UP12) is a persistent in vivo GroEL substrate (Bochkareva et al., 2002
).
Another finding supporting the role of AstRS in stationary-phase-related processes is the fact that several of the proteins that are regulated by AstR are enzymes involved in cellular energetics, as entry into the stationary phase and the induction of the anaerobic respiratory chain often go hand in hand. Enzymes important for the metabolism of aerobic organisms were decreased in the mutant (i.e. LPD, Qor and two unknown aldehyde DHs), whereas enzymes important for fermentative micro-organisms and anaerobic metabolism were increased (MoeB). LPD (lpdA gene) is an essential component of two complexes playing a crucial role in the central metabolism of aerobic organisms (pyruvate DH and 2-oxoglutarate DH). LPD is able to transfer electrons from NADH to various redox-active compounds and quinones. Excess LPD (Smith & Neidhardt, 1983) is probably involved in transport of reducing equivalents across the membrane and/or in the reduction of membrane-bound quinones (quinone redox cycling) (Owen et al., 1980
; Walker et al., 1997
; Youn & Kang, 2000
; Schwinde et al., 2001
). Qor is an electron-transport-associated component with NAD(P)H-dependent quinone redox activity. Quinones play an essential role in hydrogen transfer reactions, e.g. during aerobic and anaerobic respiration (Thorn et al., 1995
). The moeAB operon encodes proteins connecting molybdate metabolism, molybdopterin synthesis and apomolybdoenzyme synthesis during anaerobic growth (Hasona et al., 2001
). As molybdoenzymes play important roles when oxygen is limited, serving a redox function, the synthesis of Mo-cofactor is essential for optimal growth in these conditions (Wootton et al., 1991
). Interestingly, in E. coli, the transcriptional regulatory complex that induces the anaerobic respiratory chain (which includes several molybdoenzymes) during the transition to stationary phase is FlhDC (Prüß et al., 2001
).
Several other proteins (Bcp, PpiB, carbonic anhydrase, putative amidinotransferase) that may play a role in stationary-phase survival were also up-regulated in the astR background. Bcp is a probable antioxidant protein that may be important for coping with oxidative damage, especially during long-term culture. PpiB catalyses protein folding. The induction of this specific chaperone may represent an adaptation that allows protein synthesis and folding to continue during the stationary phase. This function may be particularly important in the stationary phase, when protein synthesis is drastically altered and proceeds very slowly. In B. subtilis, peptidyl-prolyl cistrans isomerases are important for growth in starvation conditions (Gothel et al., 1998). The carbonic anhydrase enzyme found in PL2106 may also be important for long-term survival. It has long been known that bacteria are dependent on the presence of CO2 for growth or for overcoming long lag times (Neidhardt et al., 1974
; Repaske & Clayton, 1978
). At least two roles have been suggested for this enzyme (Smith & Ferry, 2000
). It could provide CO2 or
for enzymic reactions or remove them to improve the energetics of the reaction. This role would be particularly important for decarboxylation reactions coupled to energy generation. In fermentative bacteria, the decarboxylation of dicarboxylic acids can serve as the sole energy source for growth. P. luminescens could also take advantage of the interconversion of a freely diffusible uncharged species (CO2) to a charged species
to perform various physiological functions such as solute transport. A possible example is the transport of acetate into the cell via a H+/acetate symport mechanism using the H+ generated from the catalysed hydration of CO2 into
. Finally, it was intriguing to find an increased level of a probable L-arginine : lysine amidinotransferase in PL2106, whereas the amount of ArgB decreased. This might signify that arginine is no longer synthesized but broken down via a new pathway to be used as source of carbon, nitrogen or energy. The identity (4850 %) found between this enzyme and L-arginine : glycine amidinotransferases of animals is in this respect highly suggestive, as these proteins are involved in creatine biosynthesis (Humm et al., 1994
).
One interesting result concerning the analysis of AstRS function is that this two-component transduction system is somewhat involved in phenotypic variation, a fairly elusive phenomenon in Photorhabdus. In this respect, the AstRS system shows functional similarities with another two-component signal transduction system studied in Pseudomonas, the GacAGacS system (Bull et al., 2001; Sanchez-Contreras et al., 2002
). In Photorhabdus, deletion of astR creates a background that seems to promote the switch to secondary cells earlier (by about 1 week) during stationary phase. The secondary phenotype is supposed to be a response to environmental conditions not favouring nematode host association. Such a decision would have to be tightly regulated, as secondary cells represent terminal and irreversible variants whose ability to reassociate with the nematode, and therefore to persist in the tripartite association, is compromised. Although appearance of variant II is naturally counter-selected by the infective juveniles in the biotope, the Photorhabdus genome probably contains regulators to control the switch. The AstRS system may be one of those. Its in vivo functions may be to ensure that the phenotypic shift occurs only when survival in the primary form is no longer likely. The shift from variant I to variant II cells increases stationary-phase cellular capacities which may be useful for survival outside usual hosts. AstR-regulated proteins may be responsible for some of these properties, as they include proteins involved in electron transport systems, energy metabolism, iron acquisition and oxidative stress. Comparative analysis of the 2D protein pattern associated with phenotypic variation adds weight to the putative role of AstRS in controlling phenotypic variation. Indeed, the relative amount of several astR-regulated proteins (i.e. UspA, Bcp, PpiB, MoeB, the carbonic anhydrase and the iron(III) ABC transporter) is similar in the wild-type secondary form to that in the primary astR mutant, being up- or down-regulated compared to the wild-type primary form (unpublished data). However, we could not exclude that AstRS may actually affect cell survival and not directly affect phenotypic variation regulation. By decreasing the survival ability of variant I cells, the astR mutation may affect their competitive advantage, resulting in a rapid takeover of the secondary variant cells under stationary-phase growth conditions. The level at which AstRS might exert its effect and the nature of the signal it perceives therefore need further investigation.
Finally, our results also suggest that stationary-phase growth, phenotypic variation and symbiosis-specific traits are linked. This is consistent with the findings of others (Bintrim & Ensign, 1998; Ciche et al., 2001
; Forst & Clarke, 2002
; Joyce & Clarke, 2003
). During the mutualistic relationship, P. luminescens is expected to require functions for attachment to the nematode intestine, nutrient acquisition or synthesis, molecular communication and resistance to host-imposed stresses, as well as regulatory mechanisms to control the expression of these functions. The identification of regulatory systems affecting the phenotypic switching, such as AstRS, may help us to understand the regulatory pathways controlling symbiosis itself and to identify factors important for symbiosis.
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ACKNOWLEDGEMENTS |
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Received 23 September 2003;
accepted 9 January 2004.
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