APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1993, p. 2511-25190099-2240/93/082511-09$02.00/0Copyright C) 1993, American Society for Microbiology

Stable Tagging of Rhizobium meliloti with the FireflyLuciferase Gene for Environmental Monitoring

ANGEL CEBOLLA, FRANCISCO RUIZ-BERRAQUERO, AND ANTONIO JOSE PALOMARES*

Departamento de Microbiologia y Parasitologia, Facultad de Farmacia,Universidad de Sevilla, 41012 Seville, Spain

Received 5 November 1992/Accepted 23 May 1993

A system for stable tagging of gram-negative bacteria with the firefly luciferase gene, luc, is described. Apreviously constructed fusion constitutively expressing luc from the APR promoter was used. Stable integrationinto the bacterial genome was achieved by use of mini-TnS delivery vectors. The procedure developed wasapplied for tagging of representative gram-negative bacteria, such as Escherichia coli, Rhizobium meliloti,Pseudomonas putida, and Agrobacterium tumefaciens. The system permitted the detection of tagged R. melilotiin the presence of more than 105 CFU per plate without the use of any selective markers (such as antibioticresistance genes). No significant differences in growth rates or soil survival were found between the markedstrain and the wild-type strain. Studies of bioluminescent R. meliloh also revealed a good correlation betweencell biomass and bioluminescence. The firefly luciferase tagging system is an easy, safe, and sensitive methodfor the detection and enumeration of bacteria in the environment.

Great emphasis has been placed on the detection andenumeration of soil bacteria released in field inoculation asan essential requirement for risk assessment. Strategies forthe detection and isolation of bacteria in environmentalsamples have traditionally been the fluorescent-antibody andselective plating techniques (for a review, see reference 13).The recent development of molecular detection techniqueshas greatly increased the ability to track microorganisms andengineered genetic markers in natural environments (for a

review, see reference 27). The molecular biology techniquesthat allow the detection of microorganisms in soil include theuse ofDNA probes (16), the polymerase chain reaction (33),the use of selective markers, such as antibiotic resistancegenes, and the use of chromogenic markers, such as ,-ga-lactosidase (9) and ,B-glucuronidase (18). None of the tech-niques mentioned above provides in situ detection in soil.DNA hybridization requires extraction of cells and removalof humid material prior to DNA extraction. For monitoringof organisms after introduction into soil, a selective markerthat does not interfere with the ability of the strain to surviveand, in the case of microorganisms that interact with plants,to promote plant growth is needed.

Luminescence-based techniques offer many of the advan-tages of the above-described techniques and add the poten-tial for in situ, nonextractive detection of marked cells in soilsamples. Luciferase systems are potential powerful tools foruse as genetic markers. One of these is that of the marinebacterium Vibrio fischeri (31), which has been used as aneffective marker (3, 19, 28). The enzyme is a dimeric proteinencoded by two genes, luxA and luxB. The eukaryoticluciferase genes, luc from the firefly Photinus pyralis (8) andthose from the luminous click beetle Pyrophorus pla-giopthalamus (34), also have been expressed successfully invarious bacteria (5, 8, 21, 25). Each gene codifies a mono-meric enzyme that catalyzes the same reaction, involvingD-luciferin, ATP, and 02. Luciferase assays are rapid andsensitive and can be done without the disruption of cells.Comparisons of kinetic parameters and quantum yields of

* Corresponding author.

luciferases point out the advantage of the firefly enzyme,which needs only 1 ATP per emitted photon, in contrast tothe requirement of 60 ATPs for the bacterial enzyme (20).Furthermore, a recent comparison of the expression of bothtypes of luciferases in Bacillus subtilis showed that bacteriaexpressing bacterial luciferase suffered a decrease in growthrate with respect to that of the parental strain (21). This factmay result in a selective disadvantage for these bacteriawhen competing with indigenous bacteria. In contrast, nosignificant variation in B. subtilis growth was observed uponexpression of eukaryotic luciferases. Furthermore, the lu-ciferase activities measured were about threefold higher thanthat of the bacterial luciferase.

Other requirements for tagging of microorganisms are thefollowing: (i) stable inheritance of the engineered tag must beensured; (ii) the risk of transferring the marker gene amongthe ecosystem populations must be avoided; (iii) the geneshould not be overexpressed; and (iv) markers conferringresistance to antibiotics should be avoided. In the presentwork, all these requirements were fulffilled by inserting theluc gene as the only marker gene into the genome of the soilbacterium Rhizobium meliloti. The tagged strains proved tobe useful for biomass measurements and for the monitoringof strain survival in soil.

MATERIALS AND METHODS

Bacterial strains and plasmids. The strains and plasmidsused in this study are described in Table 1. Escherichia coliHB101 was used to maintain and produce pKW101. Apirlysogens of E. coli can allow the maintenance of R6K suicideplasmids, because they can express the protein, absolutelyessential for R6K replication (7). Thus, for genetic manipu-lation and stable maintenance of the TnS-based deliveryplasmids, E. coli CC118 (Apir) was used. Plasmids wereintroduced into E. coli S17-1 (Xpir) by transformation; theresulting strains were then used as donors in conjugation-transposition assays.

Culture media. E. coli was maintained and grown in LB(yeast extract, 5 g/liter; tryptone, 10 g/liter; NaCl, 10 g/liter).For R meliloti, TY (yeast extract, 3 g/liter; tryptone, 5

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TABLE 1. Bacteria and plasmids

Strains and plasmids Description Reference

StrainsE. coliHB101 pro leu thi lacY endI recA hsdM Sm' A(ara-leu) araD AlacX74 galE galK 4JM109 recAl endAI gyrA96 thi hsdR17 supE44 relAl A(lac-proAB) [F' traD36 proAB Stratagene

lacIlqZAMl5]CC118 (Apir) phoA20 thi-1 rspE rpoB argE(Am) recAl; lysogenized with Xpir phage 14S17-1 (Xpir) S17-1 (32) lysogenized with Xpir phage; F- recA hsdR RP4-2 (Tc::Mu) (Km::Tn7) K. Timmis

R. meliloti1021 Wild type; Smr Nalr Nod' Fix' on alfalfa 23CR201 R. meliloti 1021 with a mini-TnS::luc insertion in the chromosome This work

A. tumefaciens B6 Wild type; Nalr; octopine-type Ti plasmid 11

P. putida 2440 hsdR derivative of strain mt-2; Nal' 29

PlasmidspKW101 Apr TcT cI857 XPR-IUC ColEl 8pAP2 Tcr XPR-lUC oriV 26pUC18Not Apr; identical to pUC18 (36) but the polylinker is flanked by NotI sites 14pNluc(PR) Ap'; XPR-IUC inserted as a HindIII fragment in pUC18Not This workpUT/mini-Tn5 Sm/Sp Apr; Sm, Spr TnS delivery plasmid 7pUTKm Apr; Kmr TnS delivery plasmid 14pTCR211 Apr; like pUT/mini-TnS Sm/Sp but with a A\PR-IUC fusion cloned in the NotI site This workpTCR241 Apr; like pUTKm but with a ?\PR-lUC fusion cloned in the NotI site This workpTCR20 Apr; like pUTKm but with a ?4PR-IUC fusion instead of the Kmr gene This work

g/liter; CaCl2, 0.84 g/liter) or TA (yeast extract, 1 g/liter;tryptone, 10 g/liter; CaCl2, 1 mM; MgSO4, 1 mM) mediumwas used. As a minimal medium for R. meliloti, we used M9minimal medium (24) with sodium succinate as a carbonsource and biotin-Mg (0.05%). Selective media were supple-mented with ampicillin (100 mg/liter), kanamycin (75 mg/liter), neomycin (50 mg/liter), nalidixic acid (15 mg/liter),spectinomycin (200 mg/liter), streptomycin (25 mg/liter), orcycloheximide (100 mg/liter).Recombinant DNA techniques. Plasmid DNA isolation,

restriction endonuclease digestion, ligation, transformation,agarose electrophoresis, and other standard recombinantDNA techniques were done by standard protocols (22).DNA linearized by endonuclease digestion was isolated withGeneclean II (Bio 101, Inc.) in accordance with the instruc-tions of the manufacturer.Chromosomal DNA from exconjugant R. meliloti strains

was prepared by a lysis procedure described by Better et al.(2). Transfer of digested DNA from agarose gels to Hy-bond-N membranes (Amersham) was carried out with Pos-siblot (Stratagene). Southern blotting against the HindIIIAPR-IUC fragment labelled with [a-32P]dCTP was done withthe multiprime DNA labelling system kit from Amersham.After hybridization, blots were washed three times with 2xSSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium cit-rate)-2% sodium dodecyl sulfate at 42°C and finally with 1xSSC at 42°C.

Construction of plasmids. Plasmid pKW101 was the source

of firefly luciferase cDNA. This plasmid contains the N-ter-minal Xcro gene codons fused in frame to the seventh codonof the luciferase gene. The translational fusion is locateddownstream of 'PR, and its expression is heat inducedbecause of the presence of the thermosensitive cI857 alleleencoding a heat-labile A repressor on pKW101. A HindIIIfragment containing only the promoter and the luc gene was

isolated and cloned into the HindIII site of pUC18Not. TheWPR-IUC DNA segment of the resulting plasmid, pNluc(PR),was isolated after NotI digestion. The NotI fragment withthe luc gene was cloned into the unique NotI site ofpUT/mini-TnS Sm/Sp and into pUTKm partially digestedwith NotI. The resulting plasmids, pTCR211 and pTCR241,respectively (Fig. 1), were selected for preliminary studies oftransposition and luciferase expression in various gram-negative bacteria. For tagging of the bacterium without theuse of antibiotic resistance genes, plasmid pTCR20 (Fig. 1)was constructed by isolating the NotI fragment of pUTKm,which lacked the KmT gene; this fragment was then ligated tothe NotI XPR-hsC fragment of pNMuc(PR). In every step, thetarget clones were screened by their ability to produce lightafter the addition of D-(-)-luciferin (see below).

Light emission measurements. For determination of theluciferase activity of intact cells in liquid cultures, 50 pl of acell suspension was placed in a tube, and 0.15 ml of 1 mMluciferin-100 mM citric acid (pH 5.0) was added. The timecourse of light emission was recorded for 1 min in anLKB1250 luminometer equipped with a chart recorder.Alternatively, for measurement of the luciferase activity incell extracts, 0.9 ml of the cell suspension was mixed with0.1 ml of buffer to yield 0.1 M potassium phosphate (pH8.0)-2 mM EDTA-1 mg of bovine serum albumin (BSA) perml-5% glycerol (final concentration). The mixture was son-icated twice on ice for 30 s each time; 0.15 ml of 25 mMglycylglycine (pH 7.8)-10 mM MgCl2-5 mM ATP-0.1 mMD-(-)-luciferin was then added to 50 ,ul of a sonicatedsample. The specific enzymatic activity was reported as thepeak height, in relative light units (RLU), relative to the cellmass estimated by measurements of the optical density at600 nm (OD6.) of the culture.

Light emission from bacterial colonies containing lucgenes was detected either by plating cells onto a nitrocellu-

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TAGGING OF R MELILOTI WITH FIREFLY LUCIFERASE

Km NtPR cro

Sm/Sp Nt

% Eluc Nt

I 1- -A

Nt

Iu ena

IuI ~--* l E - l~~~~~~~~~~~~Nt

pTCR241

pTCR211

Nt

I _ l pTCR20

FIG. 1. Scheme for the construction of suicide plasmids used for marking gram-negative bacteria. The selective markers are flanked byTnS 19-bp terminal ends (I end and 0 end) (14). The IS50R tnp gene devoid of NotI sites (tnp*) is oriented divergently from the I end.Restriction sites: E, EcoRI; Nt, NotI; Xb, XbaI.

lose filter or by blotting colonies onto filter paper. Filterswere moistened with 500 to 700 ,ul of the substrate solution(1 mM D-luciferin in 0.1 M sodium citrate [pH 5]). After adiffusion period of a few minutes, light-emitting colonieswere detected in the dark either by dark-adapted eye orphotographically with Kodak OG-1 X-ray films as describedby Wood and DeLuca (35).

Bacterial matings. E. coli S17-1 (Apir) donor strains bear-ing the transposon vectors were grown in TY medium to thelate log phase at 29°C. Recipient strains were grown in TYmedium at 29°C for 16 to 24 h (depending on the growthrate). Filters with a mixture of donor and recipient strains ina 1:4 ratio were incubated for 14 to 16 h at 29°C on thesurface of TY medium plates. Cells were resuspended in 10mM MgSO4, and the appropriate dilutions were plated on theselective medium. For donor counterselection, nalidixic acidwas added to the selective medium. Recipient cells that hadreceived the transposon marker were selected with specti-nomycin when pTCR211 was mobilized and with kanamycinor neomycin (for R meliloti) after pTCR241 mobilization.The percentages of bioluminescent recipients among thoseacquiring selective antibiotic resistance were measured.Frequencies were estimated as the ratio of recipient cellsthat had received the luminescent marker to the total numberof recipient cells.Growth experiments. Batch culturing of R meliloti was

carried out in duplicate with 250-ml Erlenmeyer flasks con-taining 100 ml of TY medium inoculated with 0.1 ml of a

stationary-phase culture. Flasks were incubated at 29°C on arotary shaker (200 rpm). Samples of 1.2 ml were removed formeasurements of absorbance and light output. The A6. ofsamples was related to cell concentration by use of standard

curves obtained for a culture ofR. meliloti 1021 grown in TYmedium.

Soil experiments. The soil used was a sandy loam (clay,15%; sand, 48%; mud, 37%; pH 7.7; total organic carbon,1.5%; conductivity of the soil extract, 368 ,uS/cm). The soilwas sterilized for 1-h periods at 120°C on 3 consecutive days.Sterile and nonsterile soils were inoculated with R melilotisuspensions in a physiological solution containing a knownamount of bacteria. Soils were incubated at 29°C. Isolationof bacterial fractions from soils and survival studies weredone as described by Hofte et al. (15). The numbers ofbacteria were estimated from viable counts. Each experi-ment was carried out in duplicate. Bacteria from soil sus-pended in a physiological solution (0.85% NaCl) were platedon TY medium. When nonsterile soil was used, R. melilotiCR201 was enumerated by use of TY medium with nalidixicacid for populations of >105/g and with both cycloheximideand streptomycin for populations of <105/g. Colonies wereblotted onto filter paper and soaked with luciferin solution,and tagged bacteria were identified by exposure to KodakOG-1 X-ray films (see above).

RESULTS

Marking gram-negative bacteria with the luc gene. Sincethe firefly luciferase gene is a eukaryotic gene, it needs to beexpressed from a prokaryotic transcriptional unit. An addi-tional, desirable feature is that the promoter used can befunctional in a broad range of bacteria. Previous works(25, 26) showed the ability of a XPR-lUC fusion to expressconstitutively high levels of luciferase in a number ofgram-negative bacteria (Pseudomonas spp., vegetative and

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2514 CEBOLLA ET AL.

symbiotic Rhizobium spp., Azotobacter vinelandii, Acineto-bacter calcoaceticus, Agrobacterium tumefaciens, andKiebsiella spp., etc.).

Gram-negative strains engineered for a variety of in situapplications must meet a number of practical requirementsfor safe and efficient performance. These include not onlythe absence of traits that may give them an advantage overnonengineered strains but also the ability to retain theacquired genotype and phenotype in the absence of selec-tion. To solve the first of these concerns, we used fireflyluciferase, since it is very specific for its substrate, D-(-)-luciferin, which is expected to be absent from all prokaryoticcells. Thus, it is very unlikely that the enzyme would providethe cells with some kind of advantage over the wild type. Forensuring the stable inheritance of the marker gene in theabsence of selective pressure, introduction of the luc geneinto the bacterial chromosome must be achieved. With theuse of the mini-TnS system developed by Herrero et al. (14),stable insertions into the bacterial chromosome were ob-tained. This transposon has proved to be functional in abroad range of bacteria (1, 6). Moreover, chromosomalinsertions of mini-Tn5 are stable because the defectiveelement does not include the transposase gene.To test the ability of the mini-TnS system to tag various

gram-negative bacteria, we applied this system to the follow-ing species, which are representative of water, soil, andplant interactions: E. coli, R. meliloti, A. tumefaciens, andPseudomonas putida. Donors were counterselected withnalidixic acid. For selection of bioluminescent transcon-jugants, kanamycin (neomycin for R. meliloti) or spectino-mycin was added to solid medium when pTCR241 or

pTCR211 was mobilized, respectively. The ability of colo-nies to produce light was also scored.

Transposition frequencies were highest for P. putida andabout 10-fold lower for R. meliloti andA. tumefaciens (Table2). We did not study other conditions to improve thefrequencies obtained, since workable transposition frequen-cies were obtained for all bacterial species tested.

Expression of bioluminescence in tagged bacteria. Althoughthe detection of light by use of a luminometer can beaccomplished even at very low levels of luciferase activity,higher levels of bioluminescence are required for the detec-tion of colonies by use of photographic films. It is thereforedesirable that luciferase expression in intact marked cells behigh enough to achieve the photographic detection of smallcolonies by standard procedures. Colonies of all taggedstrains showed adequate levels of in vivo luciferase activitiesto be photographed after 30 min of exposure. Luciferaseactivities in intact bacteria marked with the mini-TnS::lucsystem were also measured (Table 2). Similar levels of invivo luciferase activities were found for all strains. WhenpAP2, the RK2 plasmid derivative carrying a XPR-lUC fusion,was established in E. coli and R. meliloti, yields of (6.5 +0.42) x 104 and (9.9 + 0.51) x 104 RLU/OD600 unit wereobtained, respectively. These values were about 10- to15-fold higher than those obtained with mini-TnS::luc excon-jugants. Plasmids derived from RK2 are expected to bemaintained at 7.5 to 15 copies per cell (10). The lower levelsof expression obtained from the exconjugants seemed tocorrespond to a decrease in the copy number of the markergene per bacterium.

Selection of luc-tagged R. meliloti without the use of antibi-otic resistance markers. The relative high transposition fre-quencies showed by the mini-TnS delivery plasmids in R.meliloti and the total absence of a bioluminescence back-ground in the target bacteria suggested to us the possibility

TABLE 2. Transposition frequencies for luc-based transposonsand luciferase expression after insertion in the bacterial genome

DonorTro In vivo luciferaseRecipient D expression (RLU/plasmid frequency' OD6w unit)b

E. coli pTCR241 4 x 10-6 5,983 + 451

R. meliloti pTCR211 2 x 10-6 7,045 + 345pTCR241 2 x 10-6 5,755 + 583

A. tumefaciens pTCR211 1 x 10-6 8,200 + 430pTCR241 1 x 10-6 7,245 ± 305

P. putida pTCR211 5 x 10-5 6,430 + 230pTCR241 6 x 10-6 5,962 + 582

a Frequency of acquisition of the bioluminescent marker by the recipientcells after mating with E. coli S17-1 (Apir) bearing the corresponding suicideplasmid.

b Values represent the peak light intensity emitted from cultures of unbro-ken exconjugant cells at an OD60 of 0.4 to 0.6. Data are the averages ±standard deviations for at least three assays done in duplicate. The instrumentbackground was about 10 RLU.

of using the luc gene as the unique marker. For that purpose,E. coli S17-1 (Xpir) containing pTCR20 was mated with R.meliloti 1021. Taking into account that transposition fre-quencies ranged from 10-5 to 10-6, we diluted the mats in a10 mM MgSO4 solution in tubes and plated the dilutions toyield at least 105 recipient cells per plate. Plates (14-cmdiameter) of TA medium supplemented with nalidixic acidwere used to grow the recipient strain. Plates were replicaprinted onto sterile nitrocellulose filters. After 5 days ofincubation at 29°C, bacteria grown on the nitrocellulosefilters were screened for luciferase activity by photographicdetection on X-ray films. After 1 h of contact with the films,about one spot of light per plate was found (Fig. 2B).However, no light was detected on plates spread with a10-fold lower dilution. Although we could identify the zonein which the light originated, isolated colonies could not beobtained. For colony isolation, a ca. 0.5-cm-diameter patchcontaining the luminescent cells was cut from the plate andintroduced into a tube containing a physiological solution(0.84% NaCl in water). The cells were resuspended, diluted,and plated from a suitable dilution to obtain isolated colo-nies. About 1 luminescent colony per 100 total colonies wasobtained. It was significant that the light of colonies measur-ing less than 1 mm in diameter could be detected by thissystem (Fig. 2C and D). The tagged strain, R. melilotiCR201, was selected for further studies. Southern blotanalysis was used to verify the presence of the luc fusion intheR meliloti CR201 genome (Fig. 3). Hybridization signalsin genomic DNA of R. meliloti CR201 digested with PvuI orX7hoI, which does not cut within the transposon, suggested asingle insertion. No hybridization signal was found in thegenomic DNA of the wild type.

Stability of the luc tag in R. meliloti CR201. For testing ofthe maintenance of the foreign luciferase gene in the taggedbacterium, serial dilutions of mid-log-phase cultures of R.meliloti CR201 in liquid TY medium were made. After morethan 60 generations, plate counts were carried out. Biolumi-nescence was found in 400 of 400 colonies tested. In thesurvival assay of R. meliloti CR201 kept for 2 months insterile soil, every colony of more than 200 tested showedluciferase activity (see below). These observations con-firmed the predicted stability of the marker system.

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FIG. 2. Selection of an R meliloti luc-tagged strain without the use of any selective marker gene. (A) Plate containing more than iO' totalrecipients. The circled region shows the spatial localization of the luminescent Rhizobium found by X-ray film exposure. (B) Detection of aspot of light indicating insertion of the marker gene. (C) Colonies isolated from the circled region in which the spot was found. Arrowheadsindicate luc-tagged colonies identified on the X-ray film (D).

Growth in minimal and rich media. R. meliloti CR201 grewon plates of M9 minimal medium, showing that no auxotro-phic gene was disrupted by transposition. In TY medium,the growth rates of marked and unmarked R. meliloti werenot significantly different (Fig. 4). The doubling time for R.meliloti 1021 during exponential growth was 86.9 + 4.2 min;that of R. meliloti CR201 was 86.0 + 2.6 min. Thus, neitherthe marker gene insertion nor the production of fireflyluciferase caused a significant modification of the growthrate.Measurement of biomass on the basis of luminometry. Since

the expression of luciferase from '\R is constitutive, lightoutput per cell can be expected to be independent of cellconcentration and thus can be used to estimate cell concen-tration. The in vivo luminometer assay is the quickest onefor the measurement of bioluminescence. With this assay, aplot of light output and exponentially increasing cell biomasswas linear over the range investigated (Fig. 4). However,when in vivo luciferase activity was determined in thestationary phase, the correlation between biomass and RLUwas lost (data not shown). With the luciferase assay of cellextracts, maximal sensitivity could be obtained. Luciferaseactivity in extracts from mid-log-phase growing R. melilotiCR201 was (2.95 + 0.51) x 104 RLU per 108 cells. Under our

12 3 -4 5 6 7

lw 4"W 0o

7.

l.0m0,k

FIG. 3. Southern DNA hybridization with a 2-kb HindIII frag-ment of pKW101 containing 4PR-lUC as a probe. Lanes: 1, HindIII-digested X DNA (signal at the 6.6-kb fragment); 2, XhoI digestion ofpTCR20 (7.4 kb) as a positive control; 3, total DNA of R. meliloti1021 digested with XhoI; 4, total DNA ofR meliloti CR201 digestedwith PvuI; 5, total DNA of R. meliloti CR201 digested with XhoI; 6and 7, XhoI-digested total DNA from two other R meliloti strainstagged with pTCR20.

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1.4 100000

1.2

0.8

00.6~ ~ ~ ~ ~ f~ODCR201

10000

O 50000 50 100 150 200 250 300 350 400 450 500

Time (min)

FIG. 4. Evolution of cell concentrations during the exponentialgrowth of R. meliloti 1021 (0) and CR201 (-) and simultaneouschanges in the in vivo luciferase activity of CR201 (A).

experimental conditions, extrapolation suggests a lower de-tection limit of 3.4 x 104 cells.

Survival of R. meliloti CR201. As a result of study of thesurvival of CR201 and 1021 in sterile soil, it seemed that themarker gene had not been inserted in a locus important forthe viability of strain CR201. The survival of R melilotiCR201 and the wild type, 1021, in sterile soil was monitoredover a 6-week period. No significant differences were foundin the persistence of CR201 and the wild type (Fig. 5A). Bothstrains declined in number very slowly and, after 45 days,about 25% of the original bacterial number was still present.These results indicate that R. meliloti CR201 did not havethe luc gene inserted in a locus important for the survival ofthe strain. Furthermore, constitutive expression of lu-ciferase seemed not to affect survival in sterile soil. WhenCR201 and the wild-type were mixed in sterile soil, thepercentage of bioluminescent cells was approximately con-stant (Fig. SB). Thus, no significant displacement of one

A

u)

B

109

-0l

106

1io0 5 10 15 20 25 30

Time (days)35 40

O45

FIG. 5. (A) Survival ofR. meliloti 1021 (0) and CR201 (-) at theincubation temperature, 29°C, in sterile soil. Both strains were

recovered on solid TY medium. (B) Survival of a 1:1 mixture of R.meliloti 1021 and CR201 in sterile soil (0) and number of CR201 cellsas a percentage of the total cell number (0).

.~120

100

8 80 -

60 -

|40

20

80

S20

10 100 1000 10000 100000

Luminescent cells inoculated per 105 total CFUs

FIG. 6. Efficiency of detection of luminescent colonies of R.meliloti CR201 inoculated into a nonsterile soil suspension as afunction of the proportion of R. rneliloti CR201 organisms within thetotal population of colony-forming organisms. Data are averages ofat least two experiments.

strain by another was observed. The survival of the mixedstrains in sterile soil was similar to that of single populations.

Monitoring of R. meliloti CR201 in nonsterile soil. To testthe ability of the tagged strain to be monitored in a morecomplex environment, we studied the efficiency of viableluc+ cell detection in the presence of different numbers ofindigenous organisms. Dilution plate counts were carried outfor soil suspensions containing different proportions of lumi-nescent and indigenous organisms. Nonsterile soil was sus-pended in 0.85% NaCl to yield a range of soil concentrationsfrom 10 to 100 mg/ml. A culture ofR. meliloti CR201 (4 x 108cells per ml) and dilutions prepared from it were used asinocula and added to 4.5 ml of each soil suspension to yielddifferent concentrations of luc+ viable cells. Suspensionswere mixed by vortexing, and 0.1 ml was immediatelyspread on TY medium containing nalidixic acid, streptomy-cin, and cycloheximide. Luminescent and non-luminescentcolonies were enumerated after 4 days of incubation at 29°C.The indigenous viable cell concentration ranged from 2 x 106to 7 x 106 cells per g of soil. The efficiency of the enumer-ation technique is shown in Fig. 6, in which the percentage ofR. meliloti CR201 viable cells detected is plotted as afunction of the relative proportions of luminescent andindigenous microorganisms. The efficiency of enumerationdecreased with the total number of colonies per plate.Nevertheless, the detection limits were considerably broad.In the presence more than 105 microorganisms per plate,CR201 was detected, but only about 2.5% of the coloniesthat should have been on the plates were detected. About50% of cells of the inoculated strain were detected whentheir proportion ranged from 0.25 to 0.1% of the total cellpopulation. At concentrations of the tagged bacterium thatwere >1% of the total number of CFU, enumeration of theluminescent colonies was >80% efficient.We used this technique to monitor the survival of R.

meliloti CR201 in nonsterile soil (Fig. 7). Under theseconditions, the concentration of taggedR meliloti decreasedfrom about 108 to 104 CFU/g of soil in 6 weeks. The firstthree isolates from soil were plated on TY medium supple-mented with nalidixic acid (an example is shown in Fig. 8A).After 4 days of incubation on TY medium-nalidixic acidplates, 2 x 107 to 8 x 107 background colonies per g of soilwere found. Since a lower dilution of the soil samples wasneeded for detection of the tagged bacteria, a more selectivemedium was required to avoid massive occupation of the

1U1

I Il l 100

.. v _

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108

106

105

0 5 10 15 20 25 30 35 40 45 50

Time (days)FIG. 7. Survival ofR melioti CR201 at 29°C in nonsterile soil.

For the last two measurements, R meliloti CR201 was recovered onTY medium containing nalidixic acid, streptomycin, and cyclohex-imide.

plates by soil populations. We supplemented the mediumwith cycloheximide and streptomycin to allow the recoveryof 90 to 100% ofR meliloti but to prevent the growth of 90to 95% of other cultivable soil microorganisms (an exampleis shown in Fig. 8C). At 47 days after inoculation, thesamples contained about 0.62% of marked cells with respectto the total soil cell population. On the basis of the experi-ments described above, it is possible than some portions (10

to 50%) of the luminescent cells were not detected in the lastdetermination.

DISCUSSION

The firefly luciferase gene proved to be a versatile andreliable tool for tagging and monitoringR meliloti in culturesand in soil without the use of antibiotic resistance genes. Asa gene marker, the firefly luciferase gene provides a productthat can be easily detected. Although bioluminescence is nota unique phenotype in bacteria, it is very rare. In addition,both the gene and the reaction catalyzed by firefly luciferaseare absent in all known bacteria. The XPR-lUC fusion waseasily introduced into representative soil bacteria. In vivoluciferase activities were high enough to be detected photo-graphically in all strains. Levels of bioluminescence from thebacteria mutagenized with mini-Tn5::luc were equivalent.This observation may be explained because the marker geneis expected to be carried in one copy per bacterial cell.Additionally, either the efficiencies of firefly luciferase genetranscription-translation or the activities of the NPR promoterin the bacteria tested may be similar. In previous work,considerable levels of active luciferase were produced fromNPR in a large number of gram-negative bacteria (25, 26). Onthe other hand, TnS proved to be functional in many speciesof gram-negative bacteria (1, 6). It is therefore likely that the

____ B i; 00B,X, f + f~DFIG8. Plts wt irognsmfrmnntrlsoladimdecinofK eli CR01 coois()T;eimnldxcai

pltotiigasml band1we fe nclto fsi.(B htgaho uiecn ooisatrfl xoueo h ltin~paeA. (C Coone grw on. TY meiu cotiignldxcai,ccoeiie.n tetmcntiapewsotie7dyafeinclto. ()DtcinoKmeitiC21clneontepaeswnnpnlC..7

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2518 CEBOLLA ET AL.

mini-TnS::luc system will be functional in many other bac-terial species.

Since the use of antibiotic-resistant genes is undesirablefor tagging bacteria for environmental purposes, the gener-ation of luc insertions and the avoidance of those selectivemarkers might be very useful. Although the use of a secondselective marker would facilitate the identification of luchybrids, the procedure developed for obtaining R melilotiCR201 did not consume much time and provided someadvantages. Thus, the insertion of a single gene allowed us toanalyze how the luc gene would affect the genetically engi-neered microorganism, ruling out the possibility of interfer-ence with another engineered genetic construction. Forinstance, if any other gene had been introduced (such as anantibiotic resistance gene) and the growth rate of the result-ant tagged strain had been significantly affected, we wouldnot have known which gene caused that behavior.Other bacterial species studied here showed both higher

transposition frequencies than R. meliloti and similar levelsof in vivo luminescence. Therefore, the method may be usedin a way similar to that described for R. meliloti. On theother hand, new selective markers, such as resistance tobialaphos, mercuric compounds, or arsenite, have beenapplied to the mini-TnS system as alternatives to the use ofantibiotic resistance (14). The use of non-antibiotic resis-tance markers joined to luc may facilitate genetic manipula-tions and may allow the use of more selective growth mediato decrease the background colonies of an environmentalsample. However, direct attempts at tagging with pTCR20may be even quicker than the use of an additional, non-antibiotic resistance marker, whose use for each strain mustbe optimized. Also, it is desirable not to charge engineeredbacteria with many genes, and the luc system may besufficient for identifying genetically engineered microorgan-isms.

Since the transposase gene is not located within thetransposon, a foreign gene inserted into the chromosome byuse of the mini-TnS system should improve the stability ofthe classic TnS. The ends of mini-TnS transposons containvery small repeats (19 bp); therefore, the chance for recom-bination (inversion or deletion) is very low. The predictableinheritance and stability of the tag were confirmed in hun-dreds of colonies after growth in liquid medium and survivalexperiments in soil. However, for demonstrating that thelimits of the tag loss were similar to those of indigenousgenes losses, more sensitive experiments were required.We did not test whether the marker gene was inserted in

any R. meliloti megaplasmid or in the chromosome. Never-theless, both sites should guarantee the stability of theinsertion in the bacteria. Localization of the luciferase genein a plasmid may provide a versatile tool for monitoring genetransfers among soil bacteria.When the maximal growth rate in rich medium and sur-

vival in sterile soil were studied, no evidence of gain or lossof advantage over the wild-type strain were found for R.meliloti CR201. In contrast, a comparison of the expressionof bacterial and eukaryotic luciferases in B. subtilis showedgrowth delay when bacterial luciferase was produced but notwhen firefly luciferase was expressed from the same tran-scriptional unit (21).

Bacterial luciferase genes were applied to the measure-ment of bacterial biomass (28) and to the monitoring ofgenetically engineered microorganisms in soils (12, 30) andfeces (19), providing high sensitivity for bacterial detection.The efficiency of viable luminescent cell enumeration of Rmeliloti tagged with luc was superior to that described for

Erwinia carotovora marked with the bacterial luciferasegene (12). The efficiency of luminescent E. carotovoraenumeration was lower than 50% when the percentage oftagged bacteria was about 10% of the total soil population. Incontrast, when the percentage of R. meliloti CR201 wasabout 0.1% of the total CFU, a similar efficiency wasobtained. Thus, the efficiency of this method may be about100-fold higher than that previously reported.Under our standardized conditions, the cell concentration

of the luc-tagged rhizobia showed a linear relationship withthe light output. Thus, monitoring of cell growth can becarried out with simple, quick, and sensitive assays, even atlow cell densities. In addition, some of our data obtainedwith plasmids containing a constitutively expressed luc genesuggested the possibility of estimating plasmid copy numberby measuring light output. Luciferase expression from plas-mids would be compared with that obtained frommini-Tn5::luc chromosomal insertions. Potentially, in situdetection and measurement of luc-tagged bacteria may berealized by nonextractive techniques. Nevertheless, in vivolight output from bacteria in soil decreased considerably,likely because of reduced bacterial metabolism (low ATPconcentration or protein expression). The availability ofluciferase substrates (e.g., ATP and 02) may decrease whencells are under unfavorable conditions (N, 0, or C starva-tion). The measurement of luciferase expression from cellextracts with the addition of an excess of substrates may beapplied to such cases. In rhizobia, studies of competitive-ness among strains are very important for determining whichstrains can be used for inoculating legumes. Luminometrymay provide a rapid assay for measuring nodule occupancyof a luc-tagged strain versus other strains. Additionally,microbial activity also can be estimated because in vivoluminescence can decrease because of a shortage of sub-strates within the cell (e.g., ATP). In such a case, themeasurement of luminescence potential may be necessaryfor estimating cell concentration.Although acceptable sensitivity was obtained in the lumi-

nometric detection of tagged R. meliloti, the threshold oflight perception can be lowered by various approaches. (i) Amore sensitive photomultiplier tube can be used, since otherluminometers provide enhanced light detection (i.e.,LKB1251 is 10 to 30 times more sensitive than LKB1250; seereference 17). (ii) The luciferase assay can be optimized.Commercial luciferase assays have been developed(Promega Biotech); they increase about 10 times the sensi-tivity of classic assays. (iii) Stronger promoters expressingthe luciferase gene in a regulated manner, in such a way thatthe expression of the marker gene would be repressed in thehabitat of the genetically engineered microorganism butcould be sensitively detected upon induction in the labora-tory, can be used.

In summary, we show that the detection and enumerationof soil bacteria can be reliably carried out with the fireflyluciferase gene. With this system, the tagged strain, R.meliloti CR201, could be readily monitored after release tothe environment for risk assessment.

ACKNOWLEDGMENTSKenneth Timmis, Juan Luis Ramos, and Donald Helinski are

gratefully acknowledged for providing strains and plasmids. Wethank Josep Casadesus for valuable improvements in and construc-tive criticism of the manuscript. We also thank Francisco Tempranofor providing information about soil analysis.A.C. is an FPI fellowship recipient. This work was funded by

Fundaci6n Ram6n Areces (Madrid, Spain) and grants PB87-0918

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TAGGING OF R. MELILOTI WITH FIREFLY LUCIFERASE 2519

and BI090-0521 from the Comisi6n Espafiola Interministerial deCiencia y Tecnologia.

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