BIOLUMINESCENCE-BASED DETECTION OF KLEBSIELLA OXYTOCA VN13 IN THE ENVIRONMENT

The continuous expression of the Photobacterium leiognathi 54D10 lux genes coding for the bioluminescence was obtained in Klebsiella oxytoca VN13. Chromosomally and plasmid-encoded bioluminescence of strains constructed was used to monitor their survival in the barley rhizosphere during a limited field introduction in parallel with the parental strain. Lux Derivatives of R. oxytoca VN13, carrying the bioluminescence reporter plasmids survived on roots during the whole vegetative period of the plant and were not isolated from soil.


Introduction.
Monitoring of bacteria in the environment accompa nies a problem of bacteria releases.The efficiency of detection is critical in tracking the bacteria fate in natural biocenosis.At present rapid and sensitive molecular biological methods to detect bacteria in the environ ment have largely replaced conventional plating methods (see review [1]).New promising techniques based on recognition of the species/stra in specific DNA sequences have been developed recently, and might be used to monitor bacteria in environmental samples [2][3][4][5].The DNA pro bing technology enhanced by the polymerase chain reaction is sensitive and may be used for detection of nonculturable bacteria or bacteria pre sent at low density in the microbial community [6].However, results gai ned from probing analyses give information about events that took place in the past.Equally limiting is the destructive nature of new methods and their high cost.
Another approach to detection of bacteria in the environment is using of genetic markers as a tagging system for bacteria identification in a natural community.The lux genes possess the advantage over the re porter markers, such as lacZY, xylE, or gusA, because bacteria engineered to bioluminescence can be monitored during ongoing process and in nondisruptive manner.The light emission of bioluminescent bacteria can be measured rapidly and at a little cost.The sensitivity of bioluminescence measurements is comparable to detection by hybridization technique [1].
A variety of bacterial hosts have got a luminescent phenotype after introduction of the complete lux operon, and their bioluminescence has been used to monitor the process of plant-bacterial interaction [7]; to quantitate the number of specific bacteria [8,9]; to assess the survival of bacteria in the rhizosphere in environmental simulations [10]; to ob serve persistence and movement of bacteria during a limited field intro duction [11].The use of the lux genes as the marker system for longterm bacteria monitoring in the environment was discussed as problema tic because of the energy-consuming expression of the lux genes had to reduce the survival of the target cells [1].This study was undertaken for long-term monitoring of the survival of K. oxytoca VN13 in new natu ral surrounding by both traditional plating and bioluminescence-based methods.
Materials and methods.Strains and plasmids.Stra ins and plasmids used in this study are listed in Table 1.All microplot tests were performed with /(.oxytoca VN13 and its derivatives.K. oxytoca VN13 was isolated from the interior of a rice root taken from a Vietna mese rice paddy [12].The set of lux genes of Photobacterium leiognathi used to tag K. oxytoca VN13 was derived from pVG37Lux y kindly pro vided by Vladimir Gurevich (Krasnojarsk Institute of Biophysics of RAN, Russian Federation) [13].The set of seven lux genes (luxA and luxB are structural genes of the luciferase biosynthesis, luxC, luxD, and luxE encode the synthesis and recycling of aldehyde substrate, luxF and luxG are of unknown function) was cloned in pVG37Lux (personal com munication).
Two nonconjugal plasmids (pKAS18 and pMAKjOo) based on dif ferent replicons (pZE8 from Citrobacter freundii and pSCWl, respecti vely) were used to subclone the lux genes from pVG37Lux.pKASlSLux was constructed by inserting the lux genes taken as a BamHl fragment from pVG37Lux into the BamHl site of pKAS18 polylinker.The same strategy was used for construction of pMAK705Lux.pRT733Lux was con structed by inserting the lux genes taken as a BamHl fragment from pVG37Lux into the BgUI site of IS50R of TnphoA of pRT733 and trans formed into Escherichia coli SM10 lambda pir, because pRT733 and its derivative require pir in the chromosome of that strain for replication.pRT733Lux was used as a suicide vector to deliver TnphoALux to K. oxytoca VN13.The plasmid was transferred from E. coli to K. oxytoca by conjugation as described [14].Exconjugants were selected as exhibiting resistance to kanamycin (conferred by TnphoALux) and to rifampicin (conferred by the chromosome of K. oxytoca VN13).Plasmid isolation and gene cloning procedure were performed by standard methods [15].K. oxytoca VN13 was transformed by the method of Alexeyev, Gurfkovskaya [16].The stabilities in K. oxytoca VN13 of plasmids carrying the lux genes were determined by plating on nonselective medium aliquots of cultures grown up to 100 generations in the absence of selection.At least 500 colonies were screened for each aliquot.
K. oxytoca VN13 strains were grown in Luria broth and on LB agar [14].When appropriate, ampicillin or chloramphenicol were added to a concentration of 50 juig/ml and kanamycin or rifampicin to a concentrati on of 100 jug/ml.Bacteria were grown at 37 °C.
Seed inoculation and germination.An overnight li quid culture of K. oxytoca VN13 was washed and diluted to a concentra tion of 10 8 cfu/ml prior to inoculation of barley seeds.For laboratory ex periments seeds were sterilized with chloramine b (1 %).Medium [17], deficient in carbon and nitrogen sources, was used to germinate seeds.Inoculated seedlings were grown at room temperature and natural light.Randomly situated microplots (lXl m) were used in two replications for either type of bacteria tested.

Laboratory collection
Taylor et al. [22] V. Gurevich Alexeyev et al. [23] This study Hamilton et al. [24] This study Taylor et al. [22] This study Identification of bacteria reisolated from bar ley.The roots of barley inoculated with the parental strain of K. oxytoca VN13 were washed 1 hr (with shaking) with a 0.9 % solution of sodium chloride supplemented with 0.1 % tween-20, and the extract was plated on selective agar medium.Analysis was performed monthly using 3 samp-Ies of the inoculated barley.The identity of antibiotic-resistant bacteria as K. oxytoca was confirmed by fingerprinting of total proteins, as de scribed by [18].
Bioluminescence detection.Light emission by strain:-, of E. coli and K. oxytoca bearing the lux operon was detected visually in a dark room.Light emission from roots was detected by autophotography or by visual inspection after enrichment of bacteria on selective medium.Soil was removed from the roots of seedlings, and the roots were sealed in plastic Fig. 1.The colonies of the pa rental K. oxytoca VN13 strain and the luminescent K-oxytoca VN13 (pKAS18Lux) strain: light photography (a), photo graphy made in a dark room bags and exposed to RM-V X-ray film («Svema», Ukraine).The films were developed as prescribed by the manufacturer.
For a series of large-scale analyses of the barley roots sterile plastic transparent boxes with a special lid (d = 25 mm) filled with selective agar were used (Fig. 2, a, b).Different parts of the plant roots were pla ced on agar inside boxes brought to a field, and samples were incubated at 30 °C overnight.Bioluminescent bacteria grown on selective agar we re identified by inspection of the lids in a dark room.Detection of bio luminescence was performed every two weeks using 20 randomly selected barley plants for each type during May-September, 1992 and after a year, in April, 1993.Soil samples were collected for analysis in the rhizosphere of inoculated plants and between rows of barley.100 mg of soil sample was minced, diluted, and plated on selective agar.

RESULTS.
Construction of K. oxytoca strains with Lux+ phenotype.It was preferable to insert the lux genes in to chromosome of /C oxytoca VN13 where their stability might be increa sed in case of nonselective conditions in limited field experiment.We in serted the lux genes into the chromosome by transposon mutagenesis.TnphoLux was transferred into K. oxytoca VN13 by conjugation, as de scribed in Materials and Methods.A low level of bioluminescence in recombinant bacterium was obtained, and it would be difficult to detect it visually on the root system.In further experiments two new Lux+ de rivatives of K. oxytoca VN13 bearing pKAS18Lux or pMAK705Lux were constructed, and they were expected to be bright enough because of a lux genes high dosage.The plasmid-bearing strains exhibited a high le vel of bioluminescence (Fig. 1).Light emission from colonies of K. oxy toca VN13 (pKAS18Lux) and K. oxytoca VN13 (pMAK705Lux) could be observed immediately after placing plates in a dark room.Since under field conditions plasmid-bearing K. oxytoca VN13 will not be subjected to antibiotic selection, it was important to ascertain the stability of both pKAS18Lux and pMAK705Lux within K-oxytoca VN13 in the absence of selection.Results showed that while pKASl8 and pMAK705 are maintai ned without selection for at least 100 generations, their Lux+ derivatives are much less stable.In particular, the level of the lux plasmids in the population decreased to undetectable levels after 100 generations of unselected growth.and Lux+ transposant separately and placed on minimal agar in tubes.Bioluminescence was detected on the barley seedling roots in a dark room by unaided eyes, and it was concluded that the genetically modified K. oxytoca VN13 could colonize efficiently the root system of this plant.Bac teria colonizing the barley roots were reisolated on nutrient medium after 14 day since inoculation, screened on light emission, and transferred on medium supplemented with kanamycin in order to study both plasmid and transposon stability.Reisolated bacteria revealed unstability and ga ve 12 % Lux+Km r (transposant) and 59 % Lux+Km r forms (pKAS18Lux).For a field microplot test we used three variants of the recombinant Л". oxytoca VN13 marked with the lux genes and the parental strain.Bioluminescence detection in the rhizosphere of barley seedlings by autophotography was performed after the first 14 days since inoculation.Autophotographs of root infected by Lux+ transposant were obtained only af ter addition of nutrient medium to bags containing roots prepared for exposure to film.The plasmid-containing Klebsiella strains showed the pictures appeared on developed films after 2 hr exposure without addition of nutrients.Autophotographs reflected the distribution of the luminescent bacteria along the root (data not shown).Later we used cheaper techni que than autophotography which was an enrichment of bacteria on se lective agar in boxes followed by visualization of bacterial bioluminescence in darkness.The roots colonized by Lux+ transposant gave weak visible light signals on selective medium in boxes, and after 6 weeks of inoculation it.was not detected (Table 2).Lux+ derivatives of• K. oxytoca Y.N 13 bearing pKAS18Lux or pMAKLux showed a surprising survival in the barley rhizosphere during the spring-autumn period of the barley ve getation.Bioluminescent bacteria were distributed along the barley root sy'siem, as reflected Fig. 2, c.Only after harvest the population of Lux+ bacieria colonizing plants diminished, and a number of plants colonized by.bioluminescent bacteria decreased more than 2-fold in September (see Table 2).Similar results showed the study on the survival of the K. oxy toca VN13 parental strain.Attempts to detect K. oxytoca VN13 of both marked and original strains in soil samples were unsuccessful.Bacteria tested were not detected in April, 1993.
Discussion.We met a problem of bacteria monitoring in the environme J because of K. oxytoca VN13 introduction into new climate zone.Being isolated in Vietnam from the rice root along with two accompanied bacteria, K. oxytoca VN13 exhibited beneficial effect on different plants in green-house trials (unpublished data).This bacterium possesses the advantage over rhizosphere bacteria: the capability to colonize the plant root interior and use that ecological niche for recolonization of the plant surface.The stable introduction of microbes of a beneficial nature into the rhizosphere of plants has proven to be difficult because they are usu ally outcompeted by the indigenous microflora [19].We studied the sur vival of K. oxytoca VN13, bacterium with expected beneficial effect on 'the plant, during limited introduction of it to a new climate zone.The presence of bacterium on the barley roots was monitored by a conventio nal sampling method, and it was used in parallel with a bioluminescent technique in order to prove its usefullness for long-term monitoring of bac teria in the environment.The lux genes from marine bacterium Ph. leiognathi were used to construct strains of K. oxytoca VN13 with the lumi nous phenotype, and tagged bacteria were observed during period of the plant-host vegetation by means of light emitted.
Application of the lux genes for detection of bacteria in the environ mental samples normally requires the availability of instrumentaiion for the visualization and quantification of photon emission such as fiber op tics, photoncounting electronic equipment or the autophotography techni que [20].Using these devices, information on the survival, quantity, and sites of "preferential localization can be gained in a non-disruptive man ner.For the initial screening of tested bacteria in the rhizosphere it is enough to receive information whether bacteria are present in its eco logical niche or not, and to evaluate approximately a rate of the root co lonization.In this study we did not use devices for detection of biolu minescent bacteria on the plant roots but the technique of visual inspec tion of enriched bacteria present on the root samples.This technkrue re quires an availability of the Ph.leiognathi lux genes involved m synthe sis of both luciferase and its substrate, selective agar inside small boxes, a dark room, and unaided eyes.Since it is inexpensive and nonlabour-intensive, it offers the advantage to screen quickly hundreds of samples un der field conditions.It can be used for initial selection of the environ mental samples in field experiments and adapted to laboratories with a low level of instrumentation.
Using the technique of visual inspection of bioluminescent bacteria enriched on selective medium we revealed that plasmid-bearino-variants of K. oxytoca VN13 exhibited stable presence on the plant root from May \to September, 1992.Lux+ transposant had a bit poorer survival but it was observed in the barley rhizosphere during 6 weeks, and it is similar to the result obtained by J. Shaw group earler [11].The reason of poor ability to survive of K. oxytoca VN13 with chromosomally encoded bio luminescence might be explained by a transposase-mediated rearrange ments that led to unstability of the lux marker.By comparing the survi val of the parental strain with the lux plasmid-bearing ones during the plant vegetation period, we may conclude that extra genes expressed did not decrease the survival of the engineered strains in the rhizosphere.The finding that tested plasmid-bearing bacteria continuously expres sed the lux genes and survived on the barley root system during a longterm period was unexpected for us because the lux marker was unstable in continuous culture.Earlier plasmid encoded bioluminescence was em ployed for detection of bacteria in the plant rhizosphere in microcosm ex periments [7,10].Stable expression of the Vibrio fisheri lux CDABE ge nes was detected by J. Shaw and С Kado in the phytopathogenic bacte rium Xanthomonas campestris pv.campeslris 2D520 in infected cauliflo wer plant.Another results provided by group of L. de Weger demonstra ted poor survival on soybean roots of Pseudomonas fluoresceins WCS374 cells containing constitutive bioluminescence plasmid, and it was concluded by these authors that the low bioluminescence activity of cells in the rhizosphere was reduced because of a high energy demand to syn thesize the aldehyde continuously.Data concerning the bioluminescence energetics prove that luciferase does not consume ATP for oxydation of aldehyde but reduced flavin mononucleotide (FMNH 2 ), and it has to de crease a level of generation of ATP from electron transport system in cell considerably.Furthermore, fatty acid reductase does demand stochiometric quantity of ATP for production of 1 mol of aldehyde [21].Taking into account these data and evaluating results obtained by three different gro ups exploited interacted with plants bacteria we may suppose that the available plant host has to play a selective role in the survival of bioluminescent bacteria, and latter, being in a close contact with the plant may be provided with energy from the host.On the contrary, the survival abi lity in soil of bacteria tagged with the lux genes was found to be less than that in the rhizosphere [8,11].
Our results show that the endorhizosphere bacterium K. oxytoca VN13 survives and it is an active in the rhizosphere during the whole period of the host-plant vegetation.Competition of the genetically engineered klebsiella against microbes from a natural community is not too drama tic, as expected, because of a specific ecological niche.Further experi ments performed on the same model system have to show a selective ro le of the plant in the survival of endorhizosphere bacteria.
Acknowledgments.We thank V. Gurevich for pVG37Lux, B. J. J. Lugtenberg (Leiden) for discussion of a presented work, E. N. Zherebtzova (Kiev) for a critical reading manuscript, T. N. Voznyuk for tech nical assistance.This study was supported by the Ukrainian State Com mittee on Science and Technologies.

Fig. 2 .
Fig. 2. Л plastic transparent box used for enrichment of bioluminescent bacteria (а); а fragment of the barley root placed on selective agar inside a box lid (b), bioluminescence of bacteria localized on a fragment of 3.5-month old barley root and selectively en riched inside a box (exposure 20 min) (c)

Table 1
Bacterial strains and plasmids Lux Km r ori pZEB Km r ori pZEB Lux Cm r repts pSClOl Cm r repts pSCWlLux Ap T Km T TnphoA Ap r Km TnphoALux