Bailey, M.

Barr, M.

Borin, S.

Brabhu, J.

Dejonghe, W.

Dirk Van Elsas, J.

Dronen, K.

Greated, A.

Grohmann, E.

Heuer, H.

Karagouni, A.

Krogerrecklenfort, E.

Niewint, J.

Nikolakopoulou, D.

Osborn, M.

Sandaa, R.

Smalla, K.

Sobecky, P.

Tebbe, C.

Thomas, C.

Top, E.

Turner, S.

Wolters, A.

Young, J.P.W.

English

Kornelia Smalla

Ellen Krögerrecklenfort

Holger Heuer

Winnie Dejonghe

Eva Top

Mark Osborn

Jessica Niewint

Christoph Tebbe

Michelle Barr

Mark Bailey

Alicia Greated

Christopher Thomas

Sarah Turner

Peter Young

Dora Nikolakopoulou

Amalia Karagouni

Anneke Wolters

Jan Dirk van Elsas

Karine Drønen

Ruth Sandaa

Sara Borin

Julia Brabhu

Elisabeth Grohmann

Patricia Sobecky

Crossref

Microbiology

PCR-based detection of mobile genetic elements in total community DNA

SMALLA, K

KROGERRECKLENFORT, E

HEUER, H

DEJONGHE, W

Top, Eva

OSBORN, M

NIEWINT, J

TEBBE, C

BARR, M

BAILEY, M

GREATED, A

THOMAS, C

TURNER, S

YOUNG, P

NIKOLAKOPOULOU, D

KARAGOUNI, A

WOLTERS, A

VAN ELSAS, JD

DRONEN, K

SANDAA, R

BORIN, S

BRABHU, J

GROHMANN, E

SOBECKY, P

Ghent University Academic Bibliography

PCR-based detection of mobile genetic elements in total community DNA.

Top, Eva80100073877291901610483270CEC016-B25E-11E6-B104-2D2FD0AF0289

Archivsystem Ask23

Young, P.

Elsas, van, J.D.

Grohman, E.

NARCIS 

Elsas, J.D., van

Name not available

White Rose Research Online

Microbiology CommentUSs of Haemophilus or Neisseria, then theywould have been detected even if present witha moderate degree of degeneracy. If such asequence were to be non-continuous then wewould also have expected to detect the com-ponent parts as long as they were themselvessufficiently conserved.We were unaware of the papers on Acin-etobacter. However, Acinetobacter is quitedistinct from the other Gram-negative spp.with regard to DNA uptake and its very hightransformability by plasmids, and thereforelies outside of the model upon which oursearch was predicated – being perhaps moresimilar in some respects to transformationprocesses seen in Gram-positive species.Nigel J. Saunders*, John F. Peden andE. Richard MoxonMolecular Infectious Diseases Group,Institute of Molecular Medicine, Universityof Oxford, Headington, Oxford OX3 9DS,UK*For correspondence. Tel : ›44 1865222347. Fax ›44 1865 222626.e-mail : saunders!molbiol.ox.ac.ukPCR-based detection ofmobile genetic elements intotal community DNAMobile genetic elements (MGEs) endow theirhost bacteria with genetic variability andflexibility in response to environmental stress.MGEs are an important part of bacterialdiversity (8). Of the MGEs, plasmids rep-resent perhaps the most important reservoirfor both gene transfer and capture. Althoughwe have a detailed knowledge of manyplasmids, the majority of these have beenisolated from clinically important bacteriaand have been responsible for the rapid spreadof antibiotic resistance and pathogenicitydeterminants. Consequently, our knowledgeof the prevalence and diversity of plasmids inbacteria from non-clinical environments isvery limited. Systematic studies on the in-cidence and abundance of plasmids and otherMGEs in different environmental niches havenot yet been performed. Lack of informationon the distribution of MGEs in naturalenvironments is also due to the fact that onlya minor proportion of bacteria are access-ible to traditional cultivation techniques.Recently, methods for extraction of nucleicacidsdirectlyfromenvironmentalsampleshavebeen developed to allow studies of bacterialcommunities independently of cultivation.Coupled with the progress in development ofmolecular tools, this now offers a powerfulnew dimension in our ability to investigate theprevalence and diversity of MGEs in environ-mental bacteria. The application of MGE-specific primers to total community DNA cangreatly facilitate the screening of differentenvironments for the presence of specificplasmids (1). This approach allows the de-tection of MGEs independently of theculturability of their hosts, from the presenceand expression of selectablemarkers and fromtheir ability to transfer to, and replicate in, anew recipient. The main advantage of thisapproach is that large sample numbers can beanalysed, making extensive screening pro-grams for a variety of environments morerealistic.To explore the power of this technique wehave applied PCR to study the prevalence of arange of MGEs in total DNA from differentenvironments during the first workshop of theEU-fundedConcerted Action entitled ‘MobileGenetic Elements ’ Contribution to BacterialAdaptability and Diversity ’ (MECBAD;http:}}mecbad.bba.de) (8) in Braunschweig,1–6 June 1999. This workshop, organized byMark Osborn, Christoph Tebbe and KorneliaSmalla, aimed to provide a theoretical andpractical introduction to the study of theprevalence of mobile genetic elements indifferent environments.Most of the participants provided com-munity DNA from their own research pro-jects, thus allowing us to analyse a total of17 types of DNA directly extracted from:pig, chicken and cattle manure; PCB-con-taminated, uncontaminated and farm soilsamples ; oil seed rape, potato and copper-treated and untreated rhizosphere samples ;coastal salt marsh; fish farm sediment ;sewage; compost ; a waste water sample; anda linuron-degrading consortium, isolatedfrom linuron-treated soil in Belgium (seeTable 1). Amplification of 16S rRNAgenes from each environmental DNA sample,with subsequent analysis by denatur-Table 1. PCR-based detection of mobile genetic elements in total community DNA................................................................................................................................................................................................................................................................................................................................Based on Southern blot hybridization data. fi, no hybridization; (›), weak hybridization; ›, ››, ›››, hybridization, stronghybridization and very strong hybridization, respectively.Sample type IncP-1a IncP-1b IncP-9 IncQ(oriV)Tn21/Tn501-liketnpRTn21/Tn501-liketnpArepC* IntegronPCR*Compost ››› (›) fi ››› › fi fi ›››Sewage ›› ›› fi fi ››› ›› fi ›››Copper-treated rhizosphere (›) ›› (›) ››› (›) ›› (›) ›››Untreated rhizosphere fi fi fi › fi ››› (›) ›››Fish farm sediment fi › › fi ››› ››› fi fiWaste water fi fi (›) (›) ››› ››› fi ›››Farm soil fi fi fi ››› ››› ››› (›) ›››Potato rhizosphere (›) ›› fi fi › ››› ››› fiLinuron-degrading consortium from soil ››› ››› fi fi fi ›› › (›)Salt marsh (›) ›› (›) fi (›) fi fi fiUncontaminated sandy soil (A) fi fi fi fi (›) (›) fi ›PCB-contaminated sandy soil (CD2) fi › (›) fi fi › fi ›Rape rhizosphere fi fi fi fi fi fi ›› ›Mouse gut fi fi (›) fi (›) fi fi (›)Chicken manure fi fi (›) fi ››› ››› fi ›››Cattle manure fi fi (›) ››› (›) › › ›››Pig manure fi fi fi ››› ›› › › ›››* PCR products obtained.Microbiology 146, June 20001256Microbiology Commenting gradient gel electrophoresis (DGGE;3) or single-strand-conformation polymor-phism (SSCP; 6), was used to ensure thatthe DNA was of a sufficient purity (e.g. freefrom inhibitory compounds such as humicacids) for use in the subsequent PCR assaysfor MGE detection. Several primer systemsdesigned on the basis of published and un-published sequences were applied to detectIncQ (oriV) (1), IncP-1a and IncP-1b (trfA2)(1, 7), IncP-9 (rep) (2), rhizobial plasmid repCreplication regions (K. M. Palmer, S. L.Turner & J. P. W. Young, unpublished data),Tn21}Tn501-type resolvase (tnpR) (5) andtransposase (tnpA) genes (5), and gene cas-settes integrated in class I integrons (4). PCRallows the specific amplification of the DNAsequence spanned by the primers. PCR pro-ducts were analysed by agarose gel electro-phoresis and Southern blot hybridization withPCR-generated digoxigenin-labelled probesto increase the sensitivity of detection forIncQ-, IncP-1-, IncP-9-, and Tn21}Tn501-likesequences. PCR products obtained with theintegron and the repC primer were cloned andsequenced. Strong hybridization signals wereobtained for IncQ (oriV) in community DNAextracts from cattle and pig manure, in therhizosphere of copper-treated soil, and incompost and farm soil. Southern hybridiz-ation of trfA2 PCR products was performedwith both an RP4 (IncP-1a)- and an R751(IncP-1b)-generated probe. While strong hy-bridization was observed with the IncP-1b-derived probe for PCR products amplifiedfrom DNA extracted from the potato rhizo-sphere, the linuron-degrading consortium,coastal salt marsh, sewage and copper-treated soil, less intense hybridization wasobserved for compost, fish farm sedimentand PCB-contaminated soil. Hybridizationwith the IncP-1a-derived probe indicateda high prevalence of the IncP-1a-specifictrfA2 in compost, sewage and the consortiumof linuron-degrading bacteria.An interesting finding is the presence ofboth IncP-1b and IncQ (oriV) in the rhizo-sphere of the copper-treated soil, while thesereplicon-specific sequences were not detectedin the rhizosphere of the untreated soil. A lessstriking but similar observation was seen withPCB-contaminated soil (CD2), where IncP-1bplasmid-specific sequences could be detected,whereas no signal was obtained from DNA ofuncontaminated soil from the same site. IncP-9 plasmids were detected in the DNA fromvarious environmental samples, but with theexception of the fish farm sediments, weaksignals were observed. Strong hybridizationsignals, indicating a high abundance of bothTn21}Tn501-related resolvase and trans-posase genes, were detected in chicken ma-nure, sewage, waste water and farm soil,while less intense bands of the expected sizewere found for potato rhizosphere, the bio-reactor consortium (only tnpA), cattle and pigmanure, PCB-contaminated soil (tnpA only),and rhizosphere from copper-treated anduntreated soil (tnpA only for untreated soil).PCR amplification with primers flanking genecassettes integrating into class I integronsresulted in products of varying size for mostof the community DNAs. However, theseresults only indicate the presence of genecassettes. Cloning and plating on selectivemedia should allow us to detect whether theseare antibiotic resistance gene cassettes. PCRproducts amplified using the repC primerswere the expected size and surprisingly cleanfor amplifications from community DNA.The products from the eight positive repCamplifications were cloned. Positive cloneswere obtained from the potato (8}12) andoilseed rape (2}12) rhizospheres and theuntreated rhizosphere (1}12). All were pre-viously unknown repC sequence types, indi-cating previously unknown diversity in soilpopulations.Thus the workshop demonstrated that byusing PCR-based detection it is feasible toanalyse a large number of samples and toprovide data on the prevalence of MGEs.PCR screening of total community DNAallows the identification of environments thatcontain bacteria with a high incidence ofMGEs. Where proper controls exist, theapparent correlation in specific cases betweenenvironmental conditions and prevalence ofMGEs suggests that these elements may fuelresponses to selective pressure.These studies thus provide the basis forfurther work to determine whether suchpromiscuous elements carry hot spots forinsertion and whether they carry genes forphenotypic markers that are being selected inthese environments.Kornelia Smalla,1,* Ellen Kro$ gerrecklenfort,1Holger Heuer,1 Winnie Dejonghe,2 Eva Top,2Mark Osborn,3,4 Jessica Niewint,5 ChristophTebbe,5 Michelle Barr,6 Mark Bailey,6 AliciaGreated,7 Christopher Thomas,7 SarahTurner,8 Peter Young,8 DoraNikolakopoulou,9 Amalia Karagouni,9Anneke Wolters,10 Jan Dirk van Elsas,10Karine Drønen,11 Ruth Sandaa,11 SaraBorin,12,** Julia Brabhu,13 ElisabethGrohmann13,** and Patricia Sobecky14,**1Biologische Bundesanstalt fu$ r Land- undForstwirtschaft, Institut fu$ rPflanzenvirologie, Mikrobiologie undbiologische Sicherheit, Messeweg 11–12,D-38104 Braunschweig, Germany2–14University of Ghent, Belgium2; GBF,Braunschweig, Germany3; University ofEssex, UK4; FAL, Braunschweig, Germany5;NERC-Institute of Virology andEnvironmental Microbiology, Oxford, UK6;University of Birmingham, UK7; Universityof York, UK8; University of Athens, Greece9;IPO-DLO, Wageningen, The Netherlands10 ;University of Bergen, Norway11; Universityof Milano, Italy12 ; Technical UniversityBerlin, Germany13; School of BiologyGeorgia, Atlanta, USA14*For correspondence. Tel : ›49 531 2993814. Fax: ›49 531 299 3013.e-mail : k.smalla!bba.de**Guests, not members of the MECBADconsortium.1. Go$ tz, A., Pukall, R., Tietze, E., Prager, R., Tscha$ pe,H., van Elsas, J. D. & Smalla, K. (1996). Detection andcharacterization of broad-host-range plasmids inenvironmental bacteria by PCR. Appl Environ Microbiol62, 2621–2628.2. Greated, A. & Thomas, C. M. (1999). A pair of PCRprimers for IncP-9 plasmids. Microbiology 145,3003–3004.3. Heuer, H., Hartung, K., Wieland, G., Kramer, I. &Smalla, K. (1999). Polynucleotide probes that target ahypervariable region of 16S rRNA genes to identifybacterial isolates corresponding to bands of communityfingerprints. Appl Environ Microbiol 65, 1045–1049.4. Martinez-Freijo, P., Fluit, A. C., Schmitz, F. J., Grek,V. S. C., Verhoef, J. & Jones, M. E. (1998). Class Iintegrons in Gram-negative isolates from differentEuropean hospitals and association with decreasedsusceptibility to multiple antibiotic compounds.J Antimicrob Chemother 42, 689–696.5. Pearson, A. J., Bruce, K. D., Osborn, A. M., Ritchie,D. A. & Strike, P. (1996). Distribution of class IItransposase and resolvase genes in soil bacteria and theirassociation with mer genes. Appl Environ Microbiol 62,2961–2965.6. Schwieger, F. & Tebbe, C. C. (1998). A new approachto utilize PCR-single-strand-conformation polymorphismfor 16S rRNA gene-based microbial community analysis.Appl Environ Microbiol 64, 4870–4876.7. Thomas, C. M. & Thorsted, P. (1994). PCR probesfor promiscuous plasmids. Microbiology 140, 1.8. Thomas, C. M. & Smalla, K. (2000). Trawling thehorizontal gene pool. Microbiol Today 27, 24–27.Teichoic acids in bacterialcoaggregationIntergeneric and intrageneric bacterial coag-gregation are commonly observed phenom-ena, important in the growth of cultures andin the process of infection in disease (8).Intrageneric coaggregation requires divalentcations (Ca#+) and a protein, adhesin, asso-ciated with the cell surface (3). Insertionalinactivation of genes responsible for the d-alanylation of lipoteichoic acid in Strepto-coccus gordonii DL1 (Challis) has shown thatthe alanine ester residues are essential for cellaggregation (4). It was concluded that theyplay a role in providing binding sites for theputative 100 kDa adhesin and the correctpresentation or orientation of this protein forcoaggregation.In an earlier study on the teichoic acid inthe cell walls of Lactobacillus plantarumATCC 10241 and Bacillus licheniformisATCC 9945, using X-ray photoelectron spec-troscopy, it was shown that there are twodifferent forms of binding of Mg#+ to theteichoic acid (2). In the samples from whichalanine had been removed by treatment withMicrobiology 146, June 2000 1257

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PCR-based detection of mobile genetic elements in total community DNA

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