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1、Molecular Microbial Ecology ManualKluwer Academic Publishers200410.1007/1-4020-2177-1_6Section 6 - Tracking of specific microbes in the environment Lac as a marker gene to track microbes in the environmentFransDeLeij1 and NigelBainton2(1)School of Bio-medical and Life Sciences, University of Surrey,

2、 Guildford, Surrey, GU2 7XH, United Kingdom(2)School of Bio-medical and Life Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, United KingdomIntroductionThe environmental use of microorganisms as a means to combat pests and diseases, to remediate contaminated land and act as bio-fertiliser

3、s and plant growth promoters has been a focus for research for many years. However, the successful application of living microbial inocula is dependent on a thorough understanding of the behaviour of the organisms involved in relation to the environment into which they are released 8. In order to ga

4、in this type of understanding, a large variety of selective and semi-selective media have been developed that allow selective recovery of organisms from environmental samples. Such media make use of specific characteristics that are inherent to the organism under study. Such characteristics include,

5、 growth conditions (temperature, pH, etc), resistance to antibiotics and heavy metals, tolerance to osmotic pressure and/or expression of phenotypic characteristics such as fluorescent pigments, coloration and morphology. In this way, media have been designed for the isolation and quantification of

6、many ecologically important bacteria 27 Recent advances in recombinant technology have allowed the identification, characterisation and isolation of genes that encode for useful characteristics in relation to environmental applications. After isolation, and introduction into suitable vector systems,

7、 such genes can subsequently be introduced into a single recipient organism, allowing the creation of a genetically modified microorganism (GMM) with potentially improved characteristics for environmental use. However, public concerns over the possible (negative) consequences of releasing functional

8、ly improved organisms into the environment has increased the need for the development of sensitive detection techniques for microorganisms in environmental samples. The need to detect GMMs in the environment has created an impetus for the development of detection methods employing molecular techniqu

9、es. Besides genes that encode for functional characteristics that are aimed to improve the environmental performance of a GMM, a variety of genes have been isolated from several organisms to facilitate detection of a particular GMM in the environment. Depending on the type of genetic marker employed

10、, a GMM may be tracked phenotypically, relying on the expression of the marker gene by the GMM. Alternatively, the presence of a marker gene can be identified by methods that do not rely on its expression 19. In general, maker genes can be divided into three groups: Short but unique oligonucleotide

11、sequences that act as a genetic signature.Genes that provide a selectable characteristic, such as resistance to an antibiotic, a heavy metal or ability to metabolise an unusual chemical. Chromogenic markers that provide a colour change.Chromogenic markers contain one or more genes whose presence in

12、a microbial cell may be detected by an ability to produce a colour change in a substrate. Isolation of microorganisms on media supplemented with substrates that are capable of being transformed into coloured products has been used successfully to distinguish bacteria expressing the xylE gene, which

13、encodes for catechol 2,3-dioxygenase 30 and those that carry the lacZY genes from Escherichia coli. The latter encode -galactosidase (lacZ) and lactose permease (lacY) 14. Other systems in this category are GUS (expression of -glucoronidase), the lux operon from Vibrio fischeri, in which organisms a

14、re identified by their ability to bio-luminesce 28 and accumulation of GFP (Green Fluorescent protein) within cells that express the GFP genes from the jelly fish Aequorea victoria 5. The lac operon The lac operon of E. coli spans approximately 5300 base pairs and includes the lacZ, lacY and lacA ge

15、nes in addition to the operator, promoter and transcription terminator regions. Since its description 18, the lactose operon has been a model system of great usefulness in biology. Study of this operon has touched on some significant questions in biology. For example, fundamental questions of the me

16、chanisms involved in expressing genes were first studied in this system 18. The discovery of the lac repressor and its binding to an operon site on the DNA was one of the first problems concerning protein-DNA interactions to be examined 25. Studies of -galactosidase in relation to lacZ mutants have

17、been important in defining many aspects of gene-protein relationships. Studies with fragments of -galactosidase also have served as a model system for investigating protein-protein interactions. The lactose permease, the product of the second structural gene, lacY, was the first membrane transport p

18、rotein that was studied extensively. Many fundamental concepts of the transport of molecules into the cell were derived from these studies. It is therefore not surprising that structures of the lactose operon have been investigated intensively 3. For example, Fowler and Zabin 15 reported the amino-a

19、cid sequence of -galactosidase in 1978. When methods for determining DNA sequences became available, the DNA sequence of both the lac repressor and the control elements of the lactose operon was determined 3,16. After the DNA sequence of lacY was confirmed 4, the DNA sequence of lacZ was determined

20、20 with the amino acid sequence of -galactosidase as confirmation. The third component of the lac operon, lacA, was sequenced in 1985 and its amino-acid sequence determined 17. The lacA gene encodes for thiogalactosidetransacetylase and is thought to be involved in detoxification of thiogalactosides

21、 by the cell. ProceduresGenetic marking of fluorescent Pseudomonas with lacZYTo be of use for tracking microbes in the environment, marker genes need to be rare in the release environment to allow distinction between the marked organism and indigenous populations on non-selective media. Alternativel

22、y, the isolation medium should be selective, not allowing growth of indigenous bacteria that express the phenotypic features encoded by the marker gene. All fluorescent pseudomonads isolated from the environment are unable to use lactose as a carbon source. This feature makes the genes that make up

23、the lac operon ideal as genetic markers for this group of bacteria. Not only do these genes enable a recipient organism to utilise lactose as the sole carbon source, but the lacZ gene, which encodes for -galactosidase, allows cleavage of the substrate X-gal (5-chloro-4-bromo-3-indolyl-D-galactopyran

24、oside) into a bright blue product that is easily recognised 14. To determine whether lacZ alone was sufficient for lactose utilisation, lacY was eliminated on broad host-range plasmids (pMON5002 and pMON5013) which were constructed to carry genes into recipient bacteria. Plasmid pMON5002 was restric

25、ted with EcoRI at the unique site in the lacZ coding sequence and with BglII, which cuts uniquely down stream from lacY. The incorporation of the lacZ gene alone did not confer the ability of transformed Pseudomonas cells to efficiently utilise lactose as a sole carbon source. The inclusion of lacY

26、(lactose permease) enabled growth at a rate comparable to that observed on glucose. This implies that in Pseudomonas, as in E.coli, -galactosidase remains a cytoplasmic protein, which does not gain access to sufficient lactose, without an active lactose transport mechanism. The lacY product, lactose

27、 permease, provides this transport mechanism 14. Although different strategies can be employed to generate lacZY marked bacteria, the following might serve as an example of procedures employed by Barry 2 to obtain a lacZY marked P. aureofaciens that was used for release in the field 12. Although the

28、 E. coli lacZY genes expressed from different promoters on broad host-range plasmids are highly effective selectable markers for Pseudomonas 14, these genes are equally effective when delivered by a transposon Tn7-lac element into the bacterial chromosome. The advantage of Tn7 is that the transposit

29、ion gene products function in trans and only 160 base pairs at each terminus are required to allow this transposition to occur. In addition, Tn7 inserts with high specificity integrate at high frequency into the chromosomes of many Gram-negative bacteria. Tn7 typically has only one insertion site pe

30、r bacterial chromosome and is relatively rare in the environment 24. Therefore, using the Tn7 based delivery system eliminates the need for screening through unwanted transposon mutants 22. Originally the Tn7-lac element was composed of two unstable plasmids of different incompatibilities. Although

31、the method is useful for generating lac-marked Pseudomonas, features that make this method unsuitable for common use are that any manipulation of the Tn7 element has to be done on a low copy plasmid not amenable to fast multiplication 2. Also the Tn7-lac element itself contains a number of unknown r

32、egions of DNA that might originate from E. coli or from 80 or at the end of the lacA gene. The first improvement involved the use of the smaller IncQ plasmids in the cloning and delivery system. Because of the decrease in size and the apparent broader host-range of IncQ plasmids, the double IncQ sys

33、tem can be used effectively in the cloning of genes and in the introduction of Tn7-lac elements into Pseudomonas. To further facilitate the cloning steps, and in particular the construction of more versatile and more widely applicable Tn7-lac elements, a small replicon was made by making a deletion

34、of pUC8. A 500 bp fragment was cloned in this from into the E. coli chromosome containing the Tn7 insertion site. Into this E. coli strain the Tn7-lac element had been transposed and the bacterium contained a helper plasmid. From the progeny of this bacterium, a replicon of 2 kb with an 11 kb transp

35、oson was isolated. In this form the Tn7-lac element was easier to manipulate. Smaller cloning Tn7-lac elements were then constructed and their effectiveness for the expression of the lac genes determined. Mono-component Tn7-lac delivery systems were developed to expand the range of bacteria that may

36、 be marked with the lac genes. These suicide delivery vectors, based on unstable IncQ replicons or on pBRS22, eliminate the need for antibiotic sensitivities in the target bacteria and for the replication of the delivery replicon. The IncQ-based mono-component delivery system was used to mark a fluo

37、rescent pseudomonad (Ps. 3732RN) to create Ps 3732RNL11. The Tn7-lac element in Ps 3732RNL11 is composed of around 1700 bp of the termini of Tn7 and the lacZY genes (and a truncated lacA gene) promoted by the iuc operon promoter. The element used (Tn7-lac7117) contains a number of restriction sites

38、to allow ease of cloning of additional genes, promoter replacements or substitution of lac with other selectable markers. For applications in the environment, clearly stable integration of recombinant materials into the genome is desirable. Bailey et al. 1 successfully inserted two gene marker casse

39、ttes into the chromosome of a P. fluorescens isolate (SBW25). Given the potential for random and deleterious insertion, a strategy was adopted to facilitate detection whilst minimising the possibility of gene exchange and metabolic disruption. Two distinct chromosomal sites were therefore selected,

40、approximately 1Mb apart on the chromosome, namely sites Ee and site -6-. Site Ee was isolated from an EcoRI fragment from SBW25. The marker lacZY genes were inserted into a unique BglII site within the Ee site under the control of the iucA promoter isolated from pMON7117 2. This fragment was transfe

41、rred onto a mobilisable suicide delivery plasmid and integrated into the chromosome of SBW25 at the Ee site by homologous recombination. Site -6- was also isolated on an EcoRI chromosomal fragment. A marker gene cassette containing KanR and XylE genes were inserted into the unique BglII site within

42、the -6- site, with the integrating fragment being delivered by electroporation. The transformation of Pseudomonas spp. using mobilisable suicide vectors as above or electroporation as in our laboratories using pMC1871 (Pharmacia) is often difficult and inefficient, requiring large quantities of tran

43、sforming DNA and much subsequent screening. Pseudomonas fluorescens SBW25 appears to be more difficult in this regard than most other fluorescent Pseudomonas but even here methodologies have been developed for successful integration of genes. The following might serve as a guide: To provide biomass

44、suitable for transformation, careful growth of bacteria to mid exponential phase is required, using cells at OD = 0.6 (550 nm). After chilling on ice for 30 minutes, cells are pelleted by centrifugation in Falcon tubes (3K rpm for 3 minutes) followed by resuspension in 15% glycerol (V/V). Cells from

45、 an initial culture volume of 250 ml are finally resuspended in 250|I of glycerol solution and stored on ice.Purified plasmid DNA is obtained at a concentration of 1 mg/ml. Forty five ml of chilled SBW25 suspension is mixed with 5ml (5mg) of plasmid DNA in a pre-chilled microfuge tube and stored on

46、ice for 1 minute before being transferred to a pre-chilled electroporation cuvette (2 mm gap, Biorad). Electroporation is carried out using a Biorad Gene Pulsar apparatus (settings: 2.5KV, 200 (25(F).Within one minute of applying the potential, the cell suspension is mixed with 500 ml of SOB broth a

47、nd gently mixed.The contents of the cuvette is incubated at 30 C for 4 hours before plating onto tetracycline-containing LB plates followed by incubation at 30 C for 48 hours. An initial screen of recombinants is made by selecting for a TetR, KanR, lacZ+ phenotypes. Recombinants carrying the newly i

48、ntroduced DNA are identified by using PCR analysis of DNA from isolates using primers homologous to regions of DNA that should be present within the newly inserted DNA. After genotypic identification, functional assays for insertion of desired sequences are normally carried out. For example, using h

49、plc or bioassay plates where the inserted genes are responsible antibiotic biosynthesis. Integration of DNA of up to 7kb in length has been successfully achieved using this method, with predicted genotype and functionality observed. Recovery of fluorescent pseudomonads expressing lacZY from environm

50、ental samples Selective agar based mediaBacteria expressing the lacZY marker genes will be able to grow on mineral media such as M9 26 amended with 1% (w/v) lactose 14. Intrinsic resistance to antibiotics, such as rifampicin, might achieve further selection from the native microbial populations. Thi

51、s approach allows sensitive selection of up to 110 colony forming units (cfu) per g of non-sterile soil 13. Problems arise when the natural soil populations have a significant proportion of bacteria that can use lactose as a carbon source and/or is resistant to the antibiotics used to select for the

52、 recombinant strain. For example, 1% of the culturable microbial community in a silty-loam field soil (Hamble series) taken from Littlehampton (W. Sussex, UK) was able to utilise lactose as the sole carbon source. A further 3.5% of the community was resistant to kanamycin incorporated at a level of

53、100mg/l, while 0.03% of the culturable bacterial community could utilise lactose and expressed resistance to kanamycin 11. Clearly, minimal media such as M9 26, amended with lactose and/or antibiotics are of little use for the selective recovery of recombinants in such situations. In this case, it w

54、as estimated that the detection limit of a triple marked P. fluorescens strain (SBW25EeZY-6KX, expressing the lacZY genes for lactose utilisation, the aph1 gene for kanamycin resistance and the xylE gene encoding for catechol 2,3 dioxygenase), on minimal medium 26 amended with 1% (w/v) lactose, 50 m

55、g/l X-gal and 100 mg/l kanamycin, was around 103 cfu/g soil 11. On roots, where bacterial numbers are one to two log units higher than in soil, detection would only be possible if the recombinant was present in concentrations 104 cfu/g rhizosphere soil. Consequently, a more sensitive method was requ

56、ired to isolate lacZY marked Pseudomonas cells from environmental samples. A growth medium, called P-1, developed for the selective recovery of Pseudomonasstrains producing fluorescent pigment 21 provided the solution. This medium is made up as follows: KH2 PO 4 1.0 gMgSO4.7H2O 0.5 gKCl0.2 gNaNO3 5.

57、0 gDesoxycholate1.0 gBetaine5.0 gAgar15 gDistilled water1 litrepH7.27.4To obtain a clear medium, the ingredients are mixed thoroughly in a dry flask, before water is added. After mixing with water, the pH is adjusted using 1N NaOH. In our case, per flask, 5ml 1N NaOH is pipetted into a litre of medi

58、um to obtain the required pH. The medium is then autoclaved. When the medium has cooled sufficiently (4045 C), 50mg Xgal is added (1 ml stock solution of 50mg Xgal per ml DSMO). This medium will allow growth of both lacZY positive fluorescent Pseudomonas and indigenous lacZY deficient fluorescent Ps

59、eudomonas. The former will produce dark blue fluorescent colonies, while the latter will form white fluorescent colonies when viewed under long wave UV light. However, it has to be noted that the production of the blue pigment that results from the cleavage of X-gal will to some extent mask the fluo

60、rescence of the lacZY positive bacteria on agar media. The medium thus created enables phenotypic selection of the genetically marked Pseudomonas, from a background of indigenous fluorescent Pseudomonas. As indigenous fluorescent Pseudomonas are common (typically between 105106 cfu/g soil), further

61、selective agents have to be added to obtain a medium that is more selective. In the case of the triple marked P. fluorescens described before, addition of 100 mg kanamycin per litre medium, will only allow recombinants to grow. No doubt the same result can be obtained using other antibiotics to whic

62、h the strain under investigation is resistant. Surprisingly, good selective recovery can be obtained using P-1 medium from mashed root and leaf material that is colonised by fluorescent Pseudomonas, even though this material contains relative large quantities of carbohydrates and other substances th

63、at can be utilised by a variety of microorganisms. This indicates that the betaine used in the medium as a carbon source is probably not crucial for the observed selectivity for fluorescent pseudomonads. The more likely chemical in the medium that is responsible for Pseudomonas selectivity is desoxy

64、cholate. In fact, incorporation of desoxycholate into TSA (triptic soy agar) at a rate of 1g/l seems to induce selectivity for Pseudomonas (De Leij, unpublished results). Therefore, the use of selective media that obtain their selectivity independent of the carbon source allows recovery of recombina

65、nt cells from environmental samples that are relatively rich in nutrients. Examples include compost, sewage sludge, plant material and foodstuff. This also means that selective media for lacZY marked bacteria that are based on the incorporation of lactose as the sole carbon source are unlikely to remain selective when recovery is attempted from samples that contain alternative carbon sources. Most Probable Number techniques involving lacZYThe extreme selectivity of P-1 medium amended with antibiotics also allows the use