Journal of Bacteriology, March 2004, p . 1270-1279, Vol . 186, No . 5

The PhoP-PhoQ Two-Component Regulatory System of Photorhabdus luminescens Is Essential for Virulence in Insects

Sylviane Derzelle,^1* Evelyne Turlin,¹ Eric Duchaud,² Sylvie Pages,³ Frank Kunst,² Alain Givaudan,³ and Antoine Danchin¹

Unité de Génétique des Génomes Bactériens,¹ Laboratoire de Génomique des Microorganismes Pathogčnes, Institut Pasteur, 75724 Paris Cedex 15,² Laboratoire EMIP, Université Montpellier II, IFR56, Institut National de la Recherche Agronomique [UMR 1133], 34095 Montpellier Cedex 5, France³

Received 11 September 2003/ Accepted 25 November 2003

 
Photorhabdus luminescens is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ, a two-component system associated with virulence in intracellular bacterial pathogens . This organismwas shown to respond to the availability of environmental magnesium.A mutant with a knockout mutation in the regulatory componentof this system [phoP] had no obvious growth defect . It was,however, more motile and more sensitive to antimicrobial peptidesthan its wild-type parent . Remarkably, the mutation eliminatedvirulence in an insect model . No insect mortality was observedafter injection of a large number of the phoP bacteria, whilevery small amounts of parental cells killed insect larvae inless than 48 h . At the molecular level, the PhoPQ system mediatedMg²⁺-dependent modifications in lipopolysaccharides and controlleda locus [pbgPE] required for incorporation of 4-aminoarabinoseinto lipid A . Mg²⁺-regulated gene expression of pbgP1 was absentin the mutant and was restored when phoPQ was complemented intrans . This finding highlights the essential role played byPhoPQ in the virulence of an entomopathogen.

 
Pathogens have to overcome the defenses of their hosts . Photorhabdus luminescens, an insect pathogen, faces particularly challenging conditions . This bacterium has a complex life cycle that involves two completely different environments: a symbiotic stage, in which bacteria colonize the nematode gut, and a pathogenic stage,in which susceptible insects are killed by the combined actionof the nematode and the bacteria . After entering the insecthost, a nematode releases its bacterial symbionts into the insecthemocoel . Once released, the bacteria proliferate rapidly, disregardingboth the humoral insect immune response [e.g., antibacterialpeptides] and the cell-mediated insect immune response [hemocytes][14, 17] . While multiplying, the bacteria produce exo- and endotoxins,to which the insect succumbs within 48 h of infection . Theyalso produce antibiotics that inhibit the growth of competing microorganisms in the insect cadaver [16, 57] and enhance conditionsfor nematode reproduction by providing nutrients and other growthfactors utilized by the nematodes [25].

How the infecting bacteria overcome or escape from the insect immune system is still largely an open question . The recent sequencing of the strain TT01 genome revealed a plethora ofcandidate virulence factors [18] . Some of these factors have previously been found to be involved in processes that resultin insect death . The Mcf toxin [makes caterpillars floppy] was shown to be a dominant virulence factor critical for pathogenesis. The putative apoptosis action of this toxin in insect cells caused the larvae to loose body turgor and die [15] . Purified high-molecular-weight toxin complexes had both oral and injectable activities with specific effects on the midgut epithelium ofa wide range of insects [7, 70] . The protease and lipase fractionsdid not significantly affect mortality rates [8, 68], whilethe role of purified lipopolysaccharide [LPS] in virulence wasuncertain [19] . Recently, phlA, a locus encoding a hemolysinbelonging to the two-partner secretion family of proteins, wasidentified in the TT01 genome . Although PhlA appears to be producedin the hemolymph during insect infection, the high virulenceof a phlA mutant indicates that this hemolysin is not a majorvirulence determinant [9].

To colonize the nematode gut and multiply in the insect hemocoel [two environments whose physical and chemical properties differ], P . luminescens, like other bacterial pathogens [36], has evolvedtwo-component signal transduction systems [66] . These systemscomprise a membrane-associated sensor kinase and a cytoplasmictranscriptional regulator . In response to an external stimulus,the sensor component autophosphorylates at a conserved histidineresidue in an ATP-dependent reaction . In the second step, thephosphoryl group is transferred to the regulator component, promoting its binding to DNA . The two-component PhoP-PhoQ system has been found in many gram-negative bacteria [30], and itsprimary function seems to be control of physiological adaptation to Mg²⁺ availability [26] . Mg²⁺ [or Ca²⁺] binding to the periplasmicdomain of PhoQ promotes the dephosphorylation of phospho-PhoP.Expression of PhoP-activated genes is induced when the Mg²⁺ concentration is low [micromolar] and is repressed when theMg²⁺ concentration is high [millimolar] [26, 27].

In spite of its presence in both pathogenic and nonpathogenic species, PhoP-PhoQ is an important regulator of virulence genesin a number of intracellular bacterial pathogens, includingSalmonella sp., Shigella sp., Mycobacterium tuberculosis, and Neisseria meningitidis [38, 45, 46, 52] This system has been studied extensively in Salmonella enterica serovar Typhimurium, in which it regulates directly or indirectly more than 40 different genes [29, 44, 65] . Many of these genes are species specificand confer unique properties to the microorganism, includingsurvival within macrophages, resistance to host antimicrobialpeptides [APs] and acidic pH, invasion of epithelial cells,and antigen presentation [31, 45, 50, 51] . These propertiesare often linked to modifications of many components in thebacterial cell envelope governed by PhoP-PhoQ [22, 32, 43, 51].

In an attempt to identify some of the regulatory systems involved in pathogenicity control in P . luminescens, we investigated the role of the PhoP-PhoQ homologue . A knockout mutant witha mutation in the phoP regulator was generated . The mutation affected several components of the bacterial envelope and, remarkably, resulted in an avirulence phenotype in an insect model, Spodoptera littoralis . Our results suggest that PhoPQ may sense conditions in the insect hemocoel and subsequently promote resistance to the innate immunity of the insect.

 
Bacterial strains and growth conditions. Permanent stocks of all strains were maintained at -80°Cin Luria-Bertani broth supplemented with glycerol . P . luminescenswas grown at 30°C . The P . luminescens strains used wereTT01 [23] and its phoP derivative PL2104 [this study] . The Escherichia coli strains used were TG1 [59] for plasmid maintenance andS17-1 [61] for conjugation . E . coli strains were routinely grownin Luria-Bertani medium at 37°C, whereas P . luminescensstrains were grown at 30°C in Schneider medium [BioWhittaker].To study the effects of Mg²⁺ concentration, ED*-glucose definedmedium [15 mM NH₄Cl, 11 µg of ferric citrate per ml, 0.4%glucose, 2 mM K₂SO₄, 80 mM K₂HPO₄, 44 mM KH₂PO₄, 3.4 mM Na₃-citrate,50 µM FeCl₃, 7.5 µM MnCl₂, 12.5 µM ZnCl₂,2.5 µM CuCl₂, 2.5 µM CoCl₂, 2.5 µM Na₂MoO₄]containing 20 µM MgCl₂ [low-Mg²⁺ medium] or 10 mM MgCl₂[high-Mg²⁺ medium] was used for both E . coli and P . luminescens. The final concentrations of the antibiotics used for selectionwere as follows: 30 mg of gentamicin per liter, 20 mg of kanamycinper liter, and 20 mg of chloramphenicol per liter for E . coli;and 15 mg of chloramphenicol per liter for P . luminescens . All experiments were performed in accordance with the European regulation requirements concerning the contained use of group I genetically modified organisms [agreement no . 2736 CAII].

DNA manipulations and plasmid construction. Chromosomal DNA preparation, ligation, E . coli electroporation,and Southern blotting were carried out by using standard procedures[59] . Plasmid DNA was isolated with a GenElute plamid miniprepkit [Sigma] . Restriction enzymes were obtained from Roche, andenzymatic reaction products were purified with a MinElute reactioncleanup kit [Qiagen].

Plasmid pDIA604 was constructed by two steps of PCR amplification. Briefly, the chloramphenicol acetyltransferase gene of pACYC184 [Biolabs] was amplified by PCR with oligonucleotides Cat5 [5'-TTGATCGGCACGTAAGAGGT-3']and Cat3 [5'-AATTTCTGCCATTCATCCGC-3'], which resulted in an850-bp DNA fragment . Two 1.1-kb DNA fragments containing eitherthe 5' upstream region of phoP or the end of the coding regionof phoP and the downstream region were also generated by PCRby using either oligonucleotides phoP3 [5'-AAACTGCAGCTCACCGGATGGAACGCCAG-3']and phoP4 [5'-ACCTCTTACGTGCCGATCAAGGATCCGGTGTCACGAAGCTGTACC-3']or oligonucleotides phoP5 [5'-GCGGATGAATGGCAGAAATTGGATCCCGATGCTGAACTGCGCGAA-3'] and phoP6 [5'-GCTCTAGAGCGCATACTGGCACGATGCAG-3'] and genomicDNA from P . luminescens TT01 . The first 20 bases of primersphoP4 and phoP5 are complementary to primers Cat5 and Cat3,respectively . After purification with a QIAquick PCR purificationkit [Qiagen], 100-ng portions of the three previously amplifiedfragments were mixed and used as a template to generate a new3.1-kb DNA fragment by a second PCR performed with oligonucleotidesphoP3 and phoP6 . The resulting amplicon, which correspondedto a phoP::Cm fragment, was purified, restricted with PstI andXbaI, and ligated to the pJQ200KS vector [53] to obtain pDIA604.

Plasmid pDIA605 was constructed by cloning into the multiple cloning site of the pBluescript SK plasmid [Stratagene], restricted at PstI and XbaI sites, the previously amplified 3.1-kb phoP::Cm DNA fragment containing the phoP promoter region and the beginning of the phoP coding sequence.

To construct the pDIA607 plasmid, a DNA fragment containingthe whole phoPQ locus was generated by PCR with primers PhoP1 [5'-AAACTGCAGATTGAAAGCCATGACGCCAG-3'] and PhoQ2 [5'-TGCTCTAGACATCCTGAGTGTGAAGTGAA-3']. The 2.5-kb amplified fragment was purified, restricted with XbaI and PstI [underlined sites], and cloned into the pBBR1MCS-5 vector, a low-copy-number mobilizable plasmid [40] . The exactDNA sequence of pDIA606 was confirmed by sequencing [GENOME express, Montreoil, France].

Construction of a phoP mutant and complementation of the mutant. Strain PL2104 was created by allelic exchange with pDIA604 [whichcontains a cat cassette in the phoP coding region] . pDIA604was transformed into E . coli S17-1 and introduced into P . luminescensby mating . Cm^r Gm^s Sac^r exconjugants were selected on proteosepeptone agar [1% proteose peptone, 0.5% NaCl, 0.5% yeast extract,1.5% agar] containing 2% sucrose . The exconjugants had undergoneallelic exchange and lost the wild-type copy of phoP and theplasmid vehicle . Insertions were confirmed by Southern blothybridization [data not shown] by using a PCR-amplified digoxigenin-labeledphoP gene probe, oligonucleotides phoP3 and phoP4, and a PCRdigoxigenin probe synthesis kit [Roche].

Complementation was performed by using mating experiments . pDIA607 was used to transfer the phoPQ operon from E . coli S17-1 into the recipient P . luminescens PL2104 . Cm^r Gm^r exconjugants containingthe pDIA607 vector were selected.

Swimming capacity. Tryptone motility plates containing 1% Bacto Tryptone [Difco],0.5% NaCl, and 0.3% Bacto Agar [Difco] were used to test bacterialmotility as previously described [5].

In vivo pathogenicity assays. The pathogenicity assays were performed with the common cutwormS . littoralis as previously described [28] . Briefly, 20 µlof exponentially growing bacteria diluted in phosphate-bufferedsaline was injected into the hemolymph of 20 fifth-instar larvaeof S . littoralis reared on an artificial diet . The insect larvaewere then individually incubated at 23°C for up to 115 h,and bacterial CFU were determined by plating dilutions on Luria-Bertaniagar . Insect death was monitored several times after injection.Three independent experiments were performed.

Antibacterial activity. In vitro susceptibility tests to determine MICs were performedby the broth microdilution method according to National Committeefor Clinical Laboratory Standards proposed guidelines [49],with some modifications . Stock solutions of colistin methanesulfonate[Sigma] and polymyxin B [Sigma] were prepared in sterile waterto obtain concentrations of 20 and 0.5 mg/ml, respectively.Stock solutions of cecropin A and B were prepared in 0.5% aceticacid to obtain a concentration of 0.4 mg/ml . The antibioticswere then added directly to 96-well microtiter plates in twofoldserial dilutions . A total of 10⁴ CFU of bacteria that had beengrown overnight was dispensed into each microdilution well.The MICs were determined in Mueller-Hinton broth [Biokar] followingincubation at 30°C for 48 h . The microtiter plates wereread by visual observation.

RNA manipulations. Total RNA was prepared from 10-ml cultures of P . luminescensas previously described [16] . Primer extension was performedby using standard procedures [59] with some modifications, aspreviously described [16] . Briefly, 10 ng of an end-labeledprimer was annealed with total RNA, and a reverse transcriptasereaction was performed with avian myeloblastosis virus reversetranscriptase [Roche] at 42°C for 90 min . As a reference,sequencing reactions were performed with a Thermosequenase radiolabeledterminator cycle sequencing kit [Amersham] with the same primerused to map the 5' termini of phoP mRNA, phoP4 . The oligonucleotideused in primer extension experiments was end labeled with phageT4 polynucleotide kinase [BioLabs] and [-³²P]ATP [3,000 Ci/mmol]by using standard procedures [59].

LPS preparation. P . luminescens strains TT01 and PL2104 were grown to the logphase on ED*-glucose synthetic medium supplemented with MgCl₂at either a micromolar or millimolar concentration . LPS wasobtained by hot phenol-water extraction . Next, LPS extractswere mixed with 1 volume of loading buffer [Sigma] containing7% ß-mercaptoethanol and boiled for 5 min . LPS profileswere analyzed by Tricine-sodium dodecyl sulfate [SDS]-polyacrylamidegel electrophoresis [PAGE] by using a 16.5% acrylamide gel.The gel was fixed overnight in 25% isopropanol-4% acetic acidand then silver stained by using the method of Tsai and Frasch[67].

Sequence comparisons. Amino acid sequence similarity searches were carried out byusing the BLASTP software [2, 3].

Nucleotide sequence accession number. phoPQ and pbgP123E1234 sequence data have been deposited inthe EMBL databases under accession number BX470251.

 
Identification of the PhoP-PhoQ two-component system in P . luminescens. The genome sequence of P . luminescens TT01 was recently completelydeciphered [18] . Nineteen two-component regulatory systems werefound in the genome, including a counterpart of a known PhoP-PhoQsystem found in gram-negative bacteria . The organization ofthe P . luminescens phoPQ locus is similar to that of the lociin Salmonella sp . and E . coli; both genes are located downstreamof purB, encoding adenylsuccinate lyase . The sizes of the predictedP . luminescens regulator [222 residues] and sensor [487 residues]are similar to the sizes of the Salmonella and E . coli regulator[224 and 222 residues, respectively] and sensor [487 residues],and the sequences exhibit 70 and 48% identity at the amino acidlevel . In addition, the domain organization is similar . PhoPis a typical response regulator, containing an N-terminal receiverdomain and a C-terminal helix-turn-helix motif . The putativesite of phosphorylation [D58] is conserved . PhoQ contains anN-terminal periplasmic sensor domain, delineated by two hydrophobic transmembrane sequences predicted in silico [13], coupled to a cytoplasmic transmitter domain whose sequence is particularly well conserved . There are differences in the extracellular sensor domain [Fig . 1] . One interesting example of the divergence is in the ligand binding site for the divalent cations Mg²⁺ and Ca²⁺, called the acidic cluster [69] . Although the amino acidsequences were strikingly different in this cluster [DENEDNEin P . luminescens and EDDDDAE in E . coli], conservative conversionsmaintain several acidic amino acids clustered in the same region,suggesting that this region also serves as a ligand bindingsite in the P . luminescens PhoQ sensor [69].

 
Characterization and expression of the phoPQ operon from P . luminescens. phoQ starts 19 bases downstream from the coding sequence ofphoP, suggesting that the two genes form a single transcriptionunit . Primer extension analysis with total P . luminescens RNA[up to 50 µg of RNA] revealed very faint traces of phoPmRNA in exponentially growing P . luminescens cells [data notshown] . To map precisely the transcription start point, we overexpressedthe transcriptional regulatory region using plasmid pDIA607.Total RNA was extracted during the exponential growth phaseat 30°C, and primer extension analysis was performed with25 µg of RNA and primer phoP4 [Fig . 2B] . A single startpoint was mapped at an adenosine residue located 99 bp upstreamfrom the translation start codon of phoP [Fig. 2A] . This startpoint was preceded by -35 and -10 sequences [TTGCTG-17 bp-TAACAT]with significant similarity to the consensus boxes for sigma70 promoters in enterobacteria.

 
To examine the sensing function of PhoPQ in P . luminescens, the effect of the Mg²⁺ and Ca²⁺ divalent cations on phoP expressionwas investigated by primer extension . Total RNAs from P . luminescenscells transformed with plasmid pDIA607 were prepared followingexposure to various Mg²⁺ and Ca²⁺ concentrations [Fig . 2C].Expression of phoP was inversely proportional to the Mg²⁺ concentration;the maximal activation was observed in medium containing a micromolar concentration of MgCl₂ . Addition of a millimolar concentration of Mg²⁺ decreased the phoP mRNA abundance about fivefold . Growthin the presence of Ca²⁺ slightly repressed [less than twofold]phoP expression compared with expression in low-Mg²⁺medium.Thus, phoP expression in P . luminescens is controlled by a uniqueMg²⁺- and Ca²⁺-inducible promoter . It is interesting that in Salmonella and E . coli, the phoPQ operon is transcribed from two promoters [Fig . 2A], one which is active during growth inthe presence of a low concentration of Mg²⁺ and is dependenton PhoPQ and one which is constitutive [26, 39].

Construction and phenotypic characterization of a phoP mutant. To obtain clues concerning the functional role of PhoPQ in P. luminescens, the regulator PhoP was inactivated by allelic exchange. A mutant strain [PL2104] was constructed with plasmid pDIA604 harboring a chloramphenicol cassette in place of the phoP internal coding region [see Materials and Methods].

The mutant was compared to the wild-type strain in an analysisof several phenotypic traits specific to P . luminescens . Both strains adsorbed dye from nutrient bromothymol blue agar andproduced antibiotics and paracrystalline inclusion bodies atlevels indistinguishable from those produced by the wild-typestrain . The phoP mutation did not have any effect on exponential-or stationary-phase cell morphology in Schneider medium [datanot shown] . We next examined the growth characteristics of strainsTT01, PL2104, and PL2104 complemented with copies of phoPQ supplied on the low-copy-number mobilizable plasmid pDIA607 in synthetic medium containing either 10 mM or 20 µM MgCl₂ . Exceptfor a longer lag phase for strain PL2104, no significant differenceswere detected among the strains in the exponential or stationarygrowth phase [Fig . 3A] . The growth rate appeared to be only slightly lower when the magnesium level was low for PL2104.At very low concentrations of magnesium [less than 0.1 µM],all strains grew slowly and reached the stationary phase ata low cell density [optical density, 0.6 to 0.7] . At intermediatemagnesium concentrations [10 to 20 µM], all strains hadsimilar specific growth rates and reached the stationary phaseat an optical density of 1.2 to 1.5 . At both concentrations,loss of pigmentation was observed in 24-h-old cultures in allcases . At a relatively high concentration of magnesium [10 mM],all strains grew to an optical density of 5 to 5.5, and theculture broths were pigmented . These findings differ from whatwas observed with Salmonella or Neisseria; phoP mutants of theseorganisms did not grow at reduced [micromolar] magnesium levels[38, 65].

 
The first trait markedly affected by the mutation was swarmingin semisolid agar [Fig . 3B] . In 0.3% [wt/vol] agar the phoP mutant reproducibly migrated farther from the point of inoculation than the parent migrated . This was due to early onset of the spreading behavior; the mutant started to spread 15 h after inoculation, while the wild-type strain started to spread onlyafter 20 h [Fig . 3B] . Wild-type motility was restored when PL2104 was complemented with pDIA607 [data not shown].

Effect of the phoP mutation on virulence in insects. To examine the effect of the phoP mutation on virulence in insects, we injected similar doses [500 to 1,000 CFU] of parental [TT01], phoP [PL2104], and phoP-complemented [PL2104 [pDIA607]] cellsdirectly into the hemocoel of S . littoralis larvae and monitoredinsect mortality after injection [Fig . 3C] . Remarkably, no mortalitywas observed with PL2104 . Furthermore, septicemia was observed24 h after injection of wild-type bacteria, while no bacteriawere observed in the hemolymph of larvae that received the phoPmutant . This is remarkable because living cells of wild-typeP . luminescens are highly virulent when they are injected intothe hemolymph of insects . As previously reported [9], TT01 killed90% of the larvae in less than 48 h . Substantiating the roleof phoPQ, complementation with pDIA607 restored virulence, althoughwith a slight delay . This delay has two possible explanations.Some of the transformed cells may have lost the plasmid duringthe in vivo infection and therefore may have behaved like phoPmutants . Without antibiotic selection pressure, in vitro 70%of the bacterial population lost pDIA607 after 48 h of culture.Alternatively, tight regulation of phoPQ expression may be necessaryfor full virulence.

According to the definition proposed by Bucher [11], insect-pathogenicbacteria are bacteria that produce a lethal septicemia frominocula, usually less than 10,000 cells per insect . Injectionof high doses [about 10⁵ CFU] revealed that the phoP mutantis avirulent [data not shown] . Identification of the genes regulatedby PhoPQ is therefore crucial for identifying major virulencedeterminants for entomopathogenicity.

PhoP-PhoQ governed LPS modification in Mg²⁺-limited medium. The bacterial envelope is the first barrier against environmentalaggression . Since LPS is the major surface molecule and pathogenicfactor of gram-negative bacteria, a role for LPS in P . luminescensvirulence has been proposed [20] . Several gram-negative bacteriahave the ability to modify, in a PhoP-dependent manner, theirLPS in response to environmental conditions, especially in Mg²⁺-depletedmedia . The PhoPQ regulon plays a key role in the regulationof LPS production in S . enterica serovar Typhimurium and inPseudomonas aeruginosa [21, 35] and in the regulation of lipooligosaccharideproduction in Yersinia pestis [37].

Before investigating whether the LPS of P . luminescens was affected by a phoP deletion, we examined the P . luminescens LPS biosyntheticpathway in silico . To do this, BLASTP searches were performedwith the translation products of the coding sequences presentin the genome to identify putative LPS biosynthetic genes . Thisanalysis revealed that P . luminescens possesses four large locisimilar to the lpx/dnaE [lipid A], waa [LPS core], wbl [O antigen],and wec [enterobacterial common antigen] clusters found in otherEnterobacteriaceae [Fig. 4] [54] . Both the organization and the putative functions of genes found in the TT01 clusters homologous to lpx/dnaE and wec were identical to those of E . coli and S.enterica serovar Typhimurium . The organization and genes inthe waa region were more divergent, indicating that the numberand nature of the carbohydrate subunits that compose the coreoligosaccharide of the LPS are different in P . luminescens. The putative strain-specific O-antigen wbl locus of P . luminescenshas few genes homologous to those of the E . coli wbb operon.It is composed of 29 genes interrupted by a putative transposase.Of the 29 wbl genes, 9 encode proteins that are similar to sugaror UDP-sugar dehydrogenases [WblAB], epimerase [WblH], dehydratase[WblV], kinase [WblW], isomerase [WblX], phosphatase [WblZ],hydrolyase [WblK], and a homologue of the protein encoded bythe trsG gene [WblM] involved in O- antigen biosynthesis inother organisms . Three other genes encode proteins that aresimilar to amino or hexapeptide transferases [WblCDQ] . The genecluster is also predicted to code for two sugar 1-phosphate nucleotidyltransferases [WblOY] and six glycosyltransferases used for transfer of sugars to build the O unit [WblGIJFTU].Several genes were also found to be similar to genes which carryout specific assembly or processing steps during conversionof the O unit to the O antigen as part of the complete LPS,such as two putative O antigen translocase genes [wzxAB], onepotential O-antigen polymerase gene [wzy], and one gene codingfor a protein weakly similar to polymer ligase [wblL], but notto the chain length determinant gene [wzz] . Finally, the clustercontains four genes with unknown functions, including threegenes encoding putative transmembrane proteins [wblENR] . Therefore,P . luminescens likely produces an LPS consisting of three distinctstructural regions: lipid A, the core oligosaccharide, and theO-antigen polymer [strain specific or enterobacterial commonantigen].

 
The LPS was extracted from strains TT01, PL2104, and complemented PL2104 grown in low-Mg²⁺ or high-Mg²⁺ medium and was subjectedto Tricine-SDS-PAGE analysis [Fig . 5] . A comparison of the extractedLPS produced in the presence of a millimolar concentration ofMgCl₂ [Fig . 5, lanes 1 to 3] did not reveal any difference amongthe strains, either in the O-antigenic region [upper portionof the gel] or in the core-lipid A region [lower part of thegel] . In an Mg²⁺-limited environment [lanes 4 to 6], the LPSstructure in the core-lipid A region was modified, as shownby the greater intensity of the top band than of the two lowerbands . This modification was not observed in the mutant LPS[lane 5], suggesting that it was PhoP dependent . Indeed, inthe phoPQ-complemented mutant, this band was clearly overproduced[lane 6] . As the PhoPQ regulon was induced during growth ofP . luminescens in low-Mg²⁺ medium, the inability to expressthe PhoPQ regulon must have been responsible for the changeobserved in the lipid A-core structure.

 
Identification of a PhoP-dependent Mg²⁺-responsive locus involved in lipid A modification. In order to identify the possible nature of PhoP- and Mg²⁺-dependentLPS alteration in P . luminescens, a BLASTP comparison of thegenomic DNA sequence of P . luminescens with the few known Salmonella PhoP-activated genes involved in LPS modification was performed. lpxO and pagL were found to be unique to Salmonella, but homologuesof pagP and the pbgPE operon [also designated pmrHFIJKLM] wereidentified in P . luminescens . pagP encodes an outer membraneprotein responsible for incorporation of palmitate into thelipid A moiety of the LPS [55] . The seven-gene pbgPE operonmediates the synthesis of 4-aminoarabinose and incorporationof this molecule into the 4'-terminal phosphate of lipid A [33, 34] in association with pmrE [formerly pagA or ugd] [33, 47],a gene predicted to encode a UDP-glucose dehydrogenase thathas several homologues in P . luminescens . The P . luminescenspbgPE locus is predicted to contain seven genes transcribed unidirectionally, with no more than 33 bp separating any twoopen reading frames . These genes encode seven proteins whosesequences and sizes are similar to those of the Salmonella orE . coli homologues . In both S . enterica serovar Typhimuriumand E . coli, the pbgPE operon is preceded by the divergently transcribed homologous gene pmrG [previously designated pagH and ais, respectively] and is immediately followed by the divergentlytranscribed pmrD gene; both genes are thought to be PhoP regulated[58, 71] . In P . luminescens, the pbgPE operon is flanked bytwo genes that are somewhat similar to the vitamin B₁₂ transport system genes [btuCD] and by a gene similar to the gene encoding the putative lipoprotein NlpC precursor.

The transcription start site of pbgP1 and pagP was determined by primer extension, and the abundance of the corresponding mRNA was determined in strains TT01, PL2104, and PL2104[pDIA607]. P . luminescens total RNA was extracted from cells grown to the mid-log phase in ED*-glucose medium supplemented with a low concentration [20 µM] or a high concentration [10 mM]of MgCl₂ . We found that expression of both pagP [Fig . 6 and data not shown] and pbgP1 [Fig . 6] were dependent on the extracellularMg²⁺ concentration . The expression of these genes was inducedby a micromolar concentration of Mg²⁺ but was repressed by amillimolar concentration . pbgP1 expression was suppressed inthe phoP mutant strain PL2104 under both conditions . phoPQ copiessupplied by pDIA607 restored normal Mg²⁺-regulated expressionof pbgP1 in PL2104, and higher levels of expression were observedin the complemented strain . Curiously, pagP expression was not significantly affected by the phoP deletion . This indicated that PhoPQ positively controls pbgPE expression in an Mg²⁺-dependent manner, while pagP expression is induced in Mg²⁺-depleted mediumin a PhoPQ-independent manner.

 
Sensitivity to APs. phoP mutants of several intracellular pathogens are highly susceptibleto a variety of APs [30] . The PhoPQ-controlled ability to modifythe lipid A moiety of LPS is one of the main determinants thatmediate resistance to these molecules . Indeed, addition of aminoarabinoseor palmitate to lipid A in a low-Mg²⁺-concentration environmentresults in a reduction in the overall LPS negative charge, leadingto decreased binding of APs to the bacterial surface [6, 33, 22] . This prompted us to examine the sensitivity of both TT01and PL2104 to these compounds.

P . luminescens antipeptide resistance was determined by the broth microdilution method in Mueller-Hinton medium [which contained 750 µM MgCl₂ according to the manufacturer [Biokar]]. Different APs were tested [Table 1] . The phoP mutant was moresensitive to colistin, cecropins A and B, and polymyxin B thanthe wild type and the phoP-complemented mutant were . Cecropinsare considered to be the most active antimicrobial components.These small cationic peptides are naturally produced in manyinsects, including S . littoralis larvae [12].

 
A homologue of the two-component signal transduction systemPhoPQ has been identified in P . luminescens . A knockout mutant[PL2104] has many of the characteristics of other phoP mutantsof mammalian pathogens.

[i] PL2104 is more motile than its parent, strain TT01 . A similar PhoPQ-dependent effect on motility has been observed in Salmonella and P . aeruginosa . In Salmonella, decreased flagellin expressionand cell motility are coregulated by low pH and are dependenton activation of the phoPQ pathway, which directly or indirectlynegatively regulates transcription of the flagellin gene fliC[1] . Similarly, in P . aeruginosa, a phoQ mutant had a decreasedability to swim on soft agar, while a phoP null strain was considereda superswimmer [10] . Preliminary data indicate that fliC isalso up-regulated in PL2104 [unpublished data].

[ii] We demonstrated that the LPS, which comprises 40% of the outer membrane layer, is altered in a PhoP-dependent mannerwhen there is a change in the magnesium concentration . Identificationin P . luminescens of the pbgPE locus, an operon involved in synthesis of aminoarabinose and incorporation of this molecule into the lipid A moiety, and characterization of this locusas an Mg²⁺- and PhoP-dependent operon, substantiated the likelyrole played by PhoPQ in this process.

[iii] PL2104 showed enhanced sensitivity to several APs [i.e., cecropins A and B, colistin, and polymyxin] . This may have been partly correlated with its inability to perform PhoPQ-controlledLPS modification in low-Mg²⁺-concentration conditions . One mechanism of AP resistance consists of reduced electrostatic interactions between an AP and the negatively charged bacterial surface,which may occur in P . luminescens through modifications of lipidA phosphate with aminoarabinose or changes in the overall chargeof the peculiar O antigen that it harbors . Another mechanismmay involve the presence of long O-specific side chains thatsterically hinder the ability of AP to bind to the deeper partsof LPS and prevent disruption of the bacterial outer membrane.

[iv] Finally, deletion of phoP resulted in a complete loss of virulence when insects were infected with P . luminescens, while complementation restored virulence . PhoPQ-dependent defects, such as envelope alterations, greater susceptibility to insect antibacterial peptides, increased motility promoting the early recognition of flagellar components by an immune response, orthe inability to impair immune response activation through LPSsignaling, may contribute to the observed avirulence of themutant.

Surprisingly, although the primary function of PhoPQ in other gram-negative bacteria is to control physiological adaptationsin response to an Mg²⁺-limiting environment [30, 65], the P.luminescens mutant is able to grow in medium containing a lowlevel [micromolar] of magnesium without apparent difficulty.This observation might reflect a difference in control of Mg²⁺ uptake in P . luminescens . In S . enterica, three transportersmediate Mg²⁺ uptake; these are the P-type ATPases MgtA and MgtB,whose expression is transcriptionally induced in the presenceof a low Mg²⁺ concentration by PhoPQ, and the constitutive majorMg²⁺ transporter CorA [62] . Salmonella mutants defective in mgtA, mgtB, phoQ, or phoP are defective for growth in the presenceof a low Mg²⁺ concentration [65], even though CorA is expressedand functional [63] . BLASTP searches revealed little homologybetween Salmonella MgtA and MgtB and various putative P-typeATPases in P . luminescens, including a probable copper-transportingATPase [AtcU], a zinc-transporting ATPase [ZntA], and a potassium-transporting ATPase [KdpB] . In contrast, CorA, the most phylogenetically widespread Mg²⁺ transporter, is conserved . Further work is needed to determine the process of Mg²⁺ import in P . luminescens, butour analysis suggests that PhoPQ might not play a crucial role in this transport . Consistent with this hypothesis, the PhoPQ-independent pagP induction in the presence of a low Mg²⁺ concentration indicatesthat there must be additional systems that regulate gene expressionin response to an Mg²⁺-limiting environment.

As in Salmonella, phoP gene expression responds to the extracellularMg²⁺ concentration, indicating that this cation may be one ofthe physiological signals that affect the phoPQ-dependent responsein P . luminescens . phoP expression also responds to a low Ca²⁺ concentration but not to the same extent [Fig . 2C] . The promoterregion of phoP differs from that of E . coli and Salmonella. In both species, the autogenously controlled phoPQ operon is transcribed from two promoters, a PhoPQ-dependent promoter thatis active only during growth in the presence of a low Mg²⁺ concentration and another promoter that is constitutive [26, 39, 64] . Therelative positions of the constitutive and regulated promotersdiffer in the two organisms, but an authentic PhoP binding siteconsisting of a direct repeat of the heptanucleotide sequence[T]G[T]TT[AA] is conserved 25 bp upstream of the Mg²⁺-inducibletranscription start site of phoPQ [39, 72] . In P . luminescens,one Mg²⁺-inducible transcript was found under the culture conditionsused . This transcript has a long 5' untranslated region, andno obvious PhoP box is apparent upstream of its transcriptionstart point . The very faint level of phoP mRNA detected suggestedthat P . luminescens cells may be sensitive to very small changesin the amount of PhoP . It is also possible that factors presentin the insect larvae control expression of the operon . Severaloverlapping promoter-like sequences can be identified upstreamof the transcriptional start that could play a role in particularenvironments . Interestingly, the regulation of the Mg²⁺-dependentexpression of pagP and pbgPE, coding for enzymes involved inLPS modifications, also slightly differs from the regulationof the homologs in Salmonella . In Salmonella, both loci areactivated by PhoP . The control is direct for pagP but indirectfor pbgPE and occurs via PmrAB, a two-component system thatitself is induced by PhoPQ [30] . In P . luminescens, PmrAB isnot conserved, and although expression of both loci is Mg²⁺ dependent, expression of only pbgPE seems to be PhoPQ dependent.We do not know at present the regulatory mechanisms operatingin this organism for pagP and pbgPE . Nevertheless, our resultsstrongly support the hypothesis that the PhoPQ signal transductionsystem is able to respond to the Mg²⁺ content of the host environmentand transduce the signal to either induce or repress expressionof genes needed to establish an infection in insects . However,whether the Mg²⁺ concentration is directly sensed by PhoPQ orby a PhoPQ-independent mechanism which in turn regulates phoPQis still an open question.

The essential role played in vivo by PhoPQ in P . luminescens pathogenicity is somewhat unexpected for the following reasons. This system is known to control virulence, especially in intracellular pathogens, while experiments suggest that P . luminescens is extracellular in insects [60] . In Salmonella and other mammalianpathogens, the external magnesium level sensed by PhoQ is thoughtto be the signal that indicates to the organism whether it isresiding in an intracellular or extracellular compartment inthe host [29] . Mammalian extracellular fluids contain high Mg²⁺ and Ca²⁺ levels [millimolar] . Pathogens encounter low intracellularlevels [micromolar] of both cations inside macrophages or epithelialcells . Phytophagous insects, such as Spodoptera, have high Mg²⁺ and Ca²⁺ levels in the hemocoel [i.e., 33 mM Mg²⁺ and 3 mM Ca²⁺ in Spodoptera exigua] [48] . Although these concentrations shouldtheoretically repress P . luminescens phoPQ expression, inactivationof phoP prevents bacterial proliferation in the hemolymph invivo and eliminates virulence . This prompted us to make severalpredictions to account for these apparent discrepancies, notingthat compartmentalization is ubiquitous in biological processes.[i] Efficient Photorhabdus infection might start with a microenvironmentin which the Mg²⁺ concentration is particularly low . Withinminutes of its appearance in the hemolymph, the bacterium isrecognized by the insect hemocytes and encapsulated in nodules,from which it rapidly reemerges [17] . The actual mineral ionconcentrations surrounding the bacterium inside the nodulesare difficult to ascertain but might be quite different fromthose in the hemolymph . [ii] It is also possible that Photorhabdushas an intracellular phase at some point during the infection.This occurs with Y . pestis, a pathogen that is normally presentextracellularly . In this organism, PhoP is necessary early ininfection during an intracellular phase within phagocytic cells[50] . [iii] It is possible that PhoPQ is sensitive to othersignals present in vivo in the hemocoel . It has been suggestedpreviously that in Erwinia chrysanthemi and Providencia stuartii[42, 56] the PhoPQ system may sense chemical signals other thandivalent cations . In E . coli, the system has been shown to respondto a mildly acidic pH and acetate in addition to Mg²⁺ [4, 41].

In conclusion, the work described in this report showed the central role played by the phoPQ regulon in Photorhabdus virulence in insects . Further studies are required to understand how and why the PhoP mutant of P . luminescens is completely impaired in terms of virulence . Identification of the essential roleplayed in virulence by the PhoPQ signal transduction systemis an important step towards understanding how entomopathogenicbacteria such as P . luminescens perform the switch from symbiosisto pathogenicity . This finding highlights the fact that thePhoPQ regulatory system promotes pathogen resistance to hostinnate immunity in vertebrates [22] and plants [24, 42] andalso in insects.

 
We thank A . Boutonnier for helpful technical assistance withLPS preparation.

Financial support was provided by the Institut Pasteur, theCentre National de la Recherche Scientifique [URA 2171], andthe French ASG program involving Bayer CropScience, the InstitutPasteur, and INRA, supported by the Ministry of Industry.

 
* Corresponding author . Present address: INRA, Unité de Biochimie et Structure des Protéines, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France . Phone: 33 [0]1 34 65 27 66 . Fax: 33 [0]1 34 65 21 63 . E-mail: sderzell@jouy.inra.fr.

1. Adams, P., R . Fowler, N . Kinsella, G . Howell, M . Farris, P . Coote, and C . D . O'Connor. 2001 . Proteomic detection of PhoPQ- and acid-mediated repression of Salmonella motility . Proteomics 1:597-607.
2. Altschul, S . F., and D . J . Lipman. 1990 . Protein database searches for multiple alignments . Proc . Natl . Acad . Sci . 87:5509-5513.
3. Altschul, S . F., W . Gish, W . Miller, E . W . Myers, and D . J . Lipman. 1990 . Basic local alignment search tool . J . Mol . Biol . 215:403-410.
4. Bearson, B . L., L . Wilson, and J . W . Foster. 1998 . A low pH-inducible, PhoPQ-dependent acid tolerance response protects Salmonella typhimurium against inorganic acid stress . J . Bacteriol . 180:2409-2417 .
5. Bertin, P., N . Benhabiles, E . Krin, C . Laurent-Winter, C . Tendeng, E . Turlin, A . Thomas, A . Danchin, and R . Brasseur. 1999 . The structural and functional organization of H-NS-like proteins is evolutionarily conserved in Gram-negative bacteria . Mol . Microbiol . 31:319-329.
6. Bishop, R . E., H . S . Gibbons, T . Guina, M . S . Trent, S . I . Miller, and C . R . Raetz. 2000 . Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria . EMBO J . 19:5071-5080 .
7. Blackburn, M., E . Golubeva, D . Bowen, and R . H . Ffrench-Constant. 1998 . A novel insecticidal toxin from Photorhabdus luminescens, toxin complex a [Tca], and its histopathological effects on the midgut of Manduca sexta . Appl . Environ . Microbiol . 64:3036-3041 .
8. Bowen, D., M . Blackburn, T . Rocheleau, C . Grutzmacher, and R . H . Ffrench-Constant. 2000 . Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes . Insect Biochem . Mol . Biol . 30:69-74.
9. Brillard, J., E . Duchaud, N . Boemare, F . Kunst, and A . Givaudan. 2002 . The PhlA hemolysin from the entomopathogenic bacterium Photorhabdus luminescens belongs to the two-partner secretion family of hemolysins . J . Bacteriol . 184:3871-3878 .
10. Brinkman, F . S., E . L . Macfarlane, P . Warrener, and R . E . Hancock. 2001 . Evolutionary relationships among virulence-associated histidine kinases . Infect . Immun . 69:5207-5211 .
11. Bucher, G . E. 1960 . Potential bacterial pathogens of insects and their characteristics . J . Invert . Pathol . 2:172-195.
12. Choi, C . S., I . H . Lee, E . Kim, S . I . Kim, and H . R . Kim. 2000 . Antibacterial properties and partial cDNA sequences of cecropin-like antibacterial peptides from the common cutworm, Spodoptera litura . Comp . Biochem . Physiol . 125:287-297.
13. Claros, M . G., and G . von Heijne. 1994 . TopPred II: an improved software for membrane protein structure predictions . Comput . Applic . Biosci . 10:685-686.
14. 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 .
15. Daborn, P . J., N . Waterfield, C . P . Silva, C . P . Au, S . Sharma, and R . H . Ffrench-Constant. 2002 . A single Photorhabdus gene, makes caterpillars floppy [mcf], allows Escherichia coli to persist within and kill insects . Proc . Natl . Acad . Sci . 99:10742-10747 .
16. 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 .
17. Dowds, B . C . A., and A . Peters. 2002 . Virulence mechanisms, p . 79-98 . In R . Gaugler [ed.], Entomopathogenic nematology . CABI Publishing, Wallingford, United Kingdom.
18. 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 . Médigue, A . Lanois, K . Powell, P . Siguier, R . Vincent, V . Wingate, M . Zouine, P . Glaser, N . Boemare, A . Danchin, and F . Kunst. 2003 . The Photorhabdus luminescens genome reveals a biotechnological weapon to fight microbes and insect pests . Nat . Biotechnol . 21:1307-1313.
19. Dunphy, G . B. 1994 . Interaction of mutants of Xenorhabdus nematophilus [Enterobacteriaceae] with antibacterial systems of Galleria mellonella larvae [Insecta: Pyralidae] . Can . J . Microbiol . 40:161-168.
20. Dunphy, G . B., and J . M . Webster. 1988 . Virulence mechanisms of Heterorhabditids heliothidis and its bacterial associate, Xenorhabdus luminescens, in non-immune larvae of the greater wax moth, Galleria mellonela . Int . J . Parasitol . 18:729-737.
21. Ernst, R . K., E . C . Yi, L . Guo, K . B . Lim, J . L . Burns, M . Hackett, and S . Miller. 1999 . Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa . Science 286:1561-1565 .
22. Ernst, R . K., T . Guina, and S . I . Miller. 2001 . Salmonella typhimurium outer membrane remodeling: role in resistance to host innate immunity . Microbes Infect . 3:1327-1334.
23. Fischer-Le Saux, M., V . Viallard, B . Brunel, P . Normand, and N . E . Boemare. 1999 . Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P . luminescens subsp . luminescens subsp . nov., P . luminescens subsp . akhurstii subsp . nov., P . luminescens subsp . laumondii subsp . nov., P . temperata sp . nov., P . temperata subsp . temperata subsp . nov., and P . asymbiotica sp . nov . Int . J . Syst . Bacteriol . 49:1645-1656.
24. Flego, D., R . Marits, A . R . Eriksson, V . Koiv, M . B . Karlsson, R . Heikinheimo, and E . T . Palva. 2000 . A two-component regulatory system, pehR-pehS, controls endopolygalacturonase production and virulence in the plant pathogen Erwinia carotovora subsp . carotovora . Mol . Plant-Microbe Interact . 13:447-455.
25. Forst, S., and D . Clarke. 2002 . Bacteria-nematode symbiosis, p . 57-77 . In R . Gaugler [ed.], Entomopathogenic nematology . CABI Publishing, Wallingford, United Kingdom.
26. Garcia Vescovi, E., F . C . Soncini, and E . A . Groisman. 1996 . Mg²⁺ as an extracellular signal: environmental regulation of Salmonella virulence . Cell 84:165-174.
27. Garcia Vescovi, E . G., Y . M . Ayala, E . Di Cera, and E . A . Groisman. 1997 . Characterization of the bacterial sensor protein PhoQ: evidence for distinct binding sites for Mg²⁺ and Ca²⁺ . J . Biol . Chem . 272:1440-1443 .
28. Givaudan, A., and A . Lanois. 2000 . flhDC, the flagellar master operon of Xenorhabdus nematophilus: requirement for motility, lipolysis, extracellular hemolysis, and full virulence in insects . J . Bacteriol . 182:107-115 .
29. Groisman, E . A. 1998 . The ins and outs of virulence gene expression: Mg²⁺ as a regulatory signal . Bioessays 20:96-101.
30. Groisman, E . A. 2001 . The pleiotropic two-component regulatory system PhoP-PhoQ . J . Bacteriol . 183:1835-1842.
31. Groisman, E . A., C . Parra-Lopez, M . Salcedo, C . J . Lipps, and F . Heffron. 1992 . Resistance to host antimicrobial peptides is necessary for Salmonella virulence . Proc . Natl . Acad . Sci . 89:11939-11943.
32. Guina, T., E . C . Yi, H . Wang, M . Hackett, and S . I . Miller. 2000 . A PhoP-regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial peptides . J . Bacteriol . 182:4077-4086 .
33. Gunn, J . S. 2001 . Bacterial modification of LPS and resistance to antimicrobial peptides . J . Endotoxin Res . 7:57-62.
34. Gunn, J . S., K . B . Lim, J . Krueger, K . Kim, L . Guo, M . Hackett, and S . I . Miller. 1998 . Identification of PhoP-PhoQ activated genes within a duplicated region of the Salmonella typhimurium chromosome . Mol . Microbiol . 27:1171-1182.
35. Guo, L., K . B . Lim, J . S . Gunn, B . Bainbridge, R . P . Darveau, M . Hackett, and S . I . Miller. 1997 . Regulation of lipid A modifications by Salmonella typhimurium virulence genes phoP-phoQ . Science 276:250-253 .
36. Hentschel, U., M . Steinert, and J . Hacker. 2000 . Common molecular mechanisms of symbiosis and pathogenesis . Trends Microbiol . 8:226-231.
37. Hitchen, P . G., J . L . Prior, P . C . Oyston, M . Panico, B . W . Wren, R . W . Titball, H . R . Morris, and A . Dell. 2002 . Structural characterization of lipo-oligosaccharide [LOS] from Yersinia pestis: regulation of LOS structure by the PhoPQ system . Mol . Microbiol . 44:1637-1650.
38. Johnson, C . R., J . Newcombe, S . Thorne, H . A . Borde, L . J . Eales-Reynolds, A . R . Gorringe, A . R., S . G . Funnell, and J . J . McFadden. 2001 . Generation and characterization of a PhoP homologue mutant of Neisseria meningitidis . Mol . Microbiol . 39:1345-1355.
39. Kato, A., H . Tanabe, H., and R . Utsumi. 1999 . Molecular characterization of the PhoP-PhoQ two-component system in Escherichia coli K-12: identification of extracellular Mg²⁺-responsive promoters . J . Bacteriol . 181:5516-5520 .
40. Kovach, M . E., P . H . Elzer, D . S . Hill, G . T Robertson, M . A . Farris, R . M . Roop, and K . M . Peterson. 1995 . Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes . Gene 166:175-176.
41. Lesley, J . A., and C . D . Waldbuerger. 2003 . Repression of Escherichia coli PhoP-PhoQ signaling by acetate reveals a regulatory role for acetyl coenzyme A . J . Bacteriol . 185:2563-2570 .
42. Llama-Palacios, A., E . Lopez-Solanilla, C . Poza-Carrion, F . Garcia-Olmedo, and P . Rodriguez-Palenzuela. 2003 . The Erwinia chrysanthemi phoP-phoQ operon plays an important role in growth at low pH, virulence and bacterial survival in plant tissue . Mol . Microbiol . 49:347-357.
43. Macfarlane, E . L., A . Kwasnicka, M . M . Ochs, and R . E . Hancock. 1999 . PhoP-PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance . Mol . Microbiol . 34:305-316.
44. Miller, S . I., and J . J . Mekalanos. 1990 . Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages . J . Bacteriol . 172:2485-2490.
45. Miller, S . I., A . M . Kukral, and J . J . Mekalanos. 1989 . A two-component regulatory system [phoP phoQ] controls Salmonella typhimurium virulence . Proc . Natl . Acad . Sci . 86:5054-5058.
46. Moss, J . E., P . E . Fisher, B . Vick, E . A . Groisman, and A . Zychlinsky. 2000 . The regulatory protein PhoP controls susceptibility to the host inflammatory response in Shigella flexneri . Cell . Microbiol. 2:443-452.
47. Mouslim, C., and E . A . Groisman. 2003 . Control of the Salmonella ugd gene by three two-component regulatory systems . Mol . Microbiol . 47:335-344.
48. Mullins, D . E. 1985 . Chemistry and physiology of the hemolymph, p . 355-400 . In G . A . Kerkut and L . I . Gilbert [ed.], Comprehensive insect physiology, biochemistry and pharmacology . Pergamon Press, Oxford, United Kingdom.
49. National Committee for Clinical Laboratory Standards. 1997 . Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed . Approved standard M7-A4 . National Committee for Clinical laboratory Standards, Wayne, Pa.
50. Oyston, P . C., N . Dorrell, K . Williams, S . R . Li, M . Green, R . W . Titball, and B . W . Wren. 2000 . The response regulator PhoP is important for survival under conditions of macrophage-induced stress and virulence in Yersinia pestis . Infect . Immun . 68:3419-3425 .
51. Pegues, D . A., M . J . Hantman, I . Behlau, and S . I . Miller. 1995 . PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion . Mol . Microbiol . 17:169-181.
52. Perez, E., S . Samper, Y . Bordas, C . Guilhot, B . Gicquel, and C . Martin. 2001 . An essential role for phoP in Mycobacterium tuberculosis virulence . Mol . Microbiol . 41:179-187.
53. Quandt, J., and M . F . Hynes. 1993 . Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria . Gene 127:15-21.
54. Raetz, C . R. 1996 . Bacterial lipopolysaccharides: a remarkable family of bioactive macroamphiphiles, p . 1035-1063 . In F . C . Neidhardt, R . Curtiss III, J . L . Ingraham, E . C . C . Lin, K . B . Low, B . Magasanik, W . S . Reznikoff, M . Riley, M . Schaechter, and H . E . Umbarger [ed.], Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., vol . 1 . ASM Press, Washington, D.C.
55. Raetz, C . R. 2001 . Regulated covalent modifications of lipid A . J . Endotoxin Res . 7:73-78.
56. Rather, P . N., M . R . Paradise, M . M . Parojcic, and S . Patel. 1998 . A regulatory cascade involving AarG, a putative sensor kinase, controls the expression of the 2'-N-acetyltransferase and an intrinsic multiple antibiotic resistance [Mar] response in Providencia stuartii . Mol . Microbiol . 28:1345-1353.
57. 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.
58. Roland, K . L., C . R . Esther, and J . K . Spitznagel. 1994 . Isolation and characterization of a gene, pmrD, from Salmonella typhimurium that confers resistance to polymyxin when expressed in multiple copies . J . Bacteriol . 176:3589-3597.
59. Sambrook, J., E . F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual, 2nd ed . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
60. Silva, C . P., N . R . Waterfield, P . J . Daborn, P . Dean, T . Chilver, C . P . Au, S . Sharma, U . Potter, S . E . Reynolds, and R . H . Ffrench-Constant. 2002 . Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta . Cell . Microbiol . 4:329-339.
61. Simon, R. 1984 . High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon . Mol . Gen . Genet . 196:413-420.
62. Smith, R . L., M . T . Kaczmarek, L . M . Kucharski, and M . E . Maguire. 1998 . Magnesium transport in Salmonella typhimurium: regulation of mgtA and mgtCB during invasion of epithelial and macrophage cells . Microbiology 144:1835-1843.
63. Snavely, M . D., J . B . Florer, C . G . Miller, and M . E . Maguire. 1989 . Magnesium transport in Salmonella typhimurium: Mg²⁺ transport by the CorA, MgtA, and MgtB systems . J . Bacteriol . 171:4761-4766.
64. Soncini, F . C., E . Garcia Vescovi, and E . A . Groisman. 1995 . Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon . J . Bacteriol . 177:4364-4371.
65. Soncini, F . C., E . Garcia Vescovi, F . Solomon, and E . A . Groisman. 1996 . Molecular basis of the magnesium deprivation response in Salmonella typhimurium: identification of PhoP-regulated genes . J . Bacteriol . 178:5092-5099.
66. Stock, A . M., V . L . Robinson, and P . N . Goudreau. 2000 . Two-component signal transduction . Annu . Rev . Biochem . 69:183-215.
67. Tsai, C . M., and C . E . Frasch. 1982 . A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels . Anal . Biochem. 119:115-119.
68. van Sambeek, J., and A . Wiesner. 1999 . Successful parasitation of locusts by entomopathogenic nematodes is correlated with inhibition of insect phagocytes . J . Invert . Pathol . 73:154-161.
69. Waldburger, C . D., and R . T . Sauer. 1996 . Signal detection by the PhoQ sensor-transmitter . Characterization of the sensor domain and a response-impaired mutant that identifies ligand-binding determinants J . Biol . Chem . 1271:26630-26636.
70. Waterfield, N., A . Dowling, S . Sharma, P . J . Daborn, U . Potter, and R . H . ffrench-Constant. 2001 . Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli . Appl . Environ . Microbiol . 67:5017-5024 .
71. Wösten, M . M., and E . A . Groisman. 1999 . Molecular characterization of the PmrA regulon . J . Biol . Chem . 274:27185-27190 .
72. Yamamoto, K., H . Ogasawara, N . Fujita, R . Utsumi, and A . Ishihama. 2002 . Novel mode of transcription regulation of divergently overlapping promoters by PhoP, the regulator of two-component system sensing external magnesium availability . Mol . Microbiol . 45:423-438.

Free Online Full-text Article

What Is Antibiotic?, What Is Staphylococcus Aureus?, What Is Genetics?, What Is Bioreactor?, What Is Salmonella?, s, Microorganism, o, Bacteriology, o, Microorganisms, o, Microbiology, e, Bacteria, c, Escherichia coli, a, Bacillus, s, Bacillus, n, Antimicrobial, s, Microbiological, a, Streptococcal, r, Gram negative, e, Multidrug resistant, c, Escherichia coli, c, Corynebacter, e, Yeasts, c, Propionibacter, o, Antimicrobial, n, Bacillus, r, Antibiotics, i, Escherichia coli, i, Cell suspensions, o, Escherichia coli, o, Vibriosis, n, Escherichia coli, s, Agrobacterium




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005