An assessment of the potential of herbivorous insect gut bacteria to develop competence for natural transformation

Environ. Biosafety Res. 6 (2007) 135–147 c ISBR, EDP Sciences, 2007  DOI: 10.1051/ebr:2007032 Available online at: www.ebr-journal.org Thematic Issue on Horizontal Gene Transfer An assessment of the potential of herbivorous insect gut bacteria to develop competence for natural transformation Jessica L. RAY1 , Helga K. ANDERSEN1 , Sandra YOUNG2 , Kaare M. NIELSEN1,3 * and Maureen O’CALLAGHAN2 1 Department of Pharmacy, University of Tromsø, 9037 Tromsø, Norway AgResearch, P.O. Box 60, Lincoln, Canterbury, New Zealand 3 The Norwegian Institute of Gene Ecology, Science Park, 9294 Tromsø, Norway 2 Whereas the capability of DNA uptake has been well established for numerous species and strains of bacteria grown in vitro, the broader distribution of natural transformability within bacterial communities remains largely unexplored. Here, we investigate the ability of bacterial isolates from the gut of grass grub larvae (Costelytra zealandica (White); Coleoptera: Scarabaeidae) to develop natural genetic competence in vitro. A total of 37 mostly species-divergent strains isolated from the gut of grass grub larvae were selected for spontaneous rifampicin-resistance. Genomic DNA was subsequently isolated from the resistant strains and exposed to sensitive strains grown individually using established filter transformation protocols. DNA isolated from wild-type strains was used as a control. None of the 37 isolates tested exhibited a frequency of conversion to rifampicinresistance in the presence of DNA at rates that were significantly higher than the rate of spontaneous mutation to rifampicin-resistance in the presence of wild-type DNA (the limit of detection was approximately < 1 culturable transformant per 109 exposed bacteria). To further examine if conditions were conducive to bacterial DNA uptake in the grass grubs gut, we employed the competent bacterium Acinetobacter baylyi strain BD413 as a recipient species for in vivo studies. However, no transformants could be detected above the detection limit of 1 transformant per 103 cells, possibly due to low population density and limited growth of A. baylyi cells in grass grub guts. PCR analysis indicated that chromosomal Acinetobacter DNA remains detectable by PCR for up to 3 days after direct inoculation into the alimentary tract of grass grub larvae. Nevertheless, neither transforming activity of the DNA recovered from the alimentary tract of grass grubs larvae nor competence of bacterial cells recovered from inoculated larvae could be shown. Keywords: natural transformation / New Zealand grass grub / Acinetobacter / DNA uptake / DNA persistence / GMO / biosafety INTRODUCTION Large-scale usage of genetically modified organisms (GMOs) in agriculture has raised concerns over the potential for transgenes to be horizontally acquired by representatives of various exposed microbial communities (Deni et al., 2005; Gebhard and Smalla, 1999; Nielsen et al., 1998; 2005; Paget et al., 1998). More than 10 different studies now show that if high DNA sequence similarity is present between the GMOs and the recipient bacterium, recombination of transgenes into the genome of naturally competent bacteria occurs at detectable frequencies in vitro (de Vries and Wackernagel, 1998; de Vries et al., 2001; 2004; Gebhard and Smalla, 1998; Nielsen et al., 2000b; Tepfer et al., 2003), in ster* Corresponding author: ile soil (Nielsen et al., 2000b), or in infected tobacco plants (Kay et al., 2002a; 2002b). Without an introduced DNA sequence similarity, no studies have shown uptake of plant transgenes into exposed bacteria (Broer et al., 1996; Nielsen et al., 1997c; Schlüter et al., 1995). The extent to which natural homologies between transgenes and competent bacteria exist is unclear, but they may be prevalent, since most plant transgenes and vector sequences are modified from bacterial origins (Bensasson et al., 2004). The conditions promoting competence development in many bacterial strains and species have been described (de Vries and Wackernagel, 2004; Lorenz and Wackernagel, 1994). However, few studies have determined the broader distribution of natural competence in a wider range of bacterial communities (Cohan et al., 1991; Sikorski et al., 2002; Stewart and Sinigalliano, Article published by EDP Sciences and available at http://www.ebr-journal.org or http://dx.doi.org/10.1051/ebr:2007032 J.L. Ray et al. Figure 1. New Zealand grass grub (Costelytra zealandica (White)) (Coleoptera: Scarabaeidae: Melolonthinae). (a) Healthy specimen of the third instar larval stage. Note the darkly colored lumen contents beneath the white cuticle. (b) Dissected grass grub larval alimentary tract, showing the head, foregut, midgut, hindgut and fermentation sac. Figure adapted with permission from Hurst and Jackson (2002). 1990). Here we examine the transformability of a range of bacterial species obtained from within the gut of the herbivorous grass grub larva (Costelytra zealandica; Coleoptera: Scarabiaedae) from New Zealand. All of the 37 bacterial isolates characterized are thus representatives of an environment hypothesized to encounter exposure to nuclear and organelle DNA released from mechanically disrupted and ingested plant material in the insect gut (see review by Nielsen et al., this issue). We also present more detailed studies of whether conditions are conducive for in vivo transformation of the Acinetobacter baylyi strain BD413 in the gut of the larvae of the New Zealand grass grub. Strain BD413 is naturally competent during growth, and has often been used for transformation studies in vitro (Averhoff et al., 1992; de Vries and Wackernagel, 2002; Juni, 1972; Juni and Janik, 1969; Palmen and Hellingwerf, 1997), in soil and water microcosms (Chamier et al., 1993; Clerc and Simonet, 1998; Lorenz et al., 1992; Nielsen and van Elsas, 2001; Nielsen et al., 1997a; 1997b), in a river (Williams et al., 1996), in planta (Kay et al., 2002a; 2002b; Tepfer et al., 2003) and most recently in vivo in tobacco horn worm (Deni et al., 2005). Strain DB413 was originally isolated from soil. Due to the high numbers of bacterial cells, an abundance of nutrients and constant supply of DNA from ingested food material and from the death of inhabiting microbes, the insect gut is an attractive location to study the potential for in vivo transformation (Deni et al., 2005; Mohr and Tebbe, 2006). The New Zealand grass grub is an agricultural pest that feeds on roots of pasture plants during its larval stages. It is endemic to New Zealand but has adapted to feeding intensively on the roots of introduced pasture and crop plants (Ferro, 1976). The soil-dwelling larvae feed extensively on the grasses and clover in New Zealand’s improved pastures, causing significant economic damage. 136 At present there are no GM grasses or clover grown in the field in New Zealand, but GM varieties are under development both in New Zealand (McManus et al., 2005) and overseas (Wang and Ge, (...truncated)