COBISS: 1.01 Agris category code: /
ISOLATION AND uSE OF Prevotella ruminicola TC18 pLASMID pTC18 In Escherichia coli-P. ruminicola
shuttle vector construction
Tomaž ACCETTO
delo je prispelo 13. marca 2009, sprejeto 03. junija 2009. Received March 13, 2009; accepted June 03, 2009.
Isolation and use of Prevotella ruminicola TC18 plasmid pTC18 in Escherichia coli-P. ruminicola shuttle vector construction
A cryptic plasmid of approximately 3 kilobases named pTC18 was discovered in a ruminal Prevotella ruminicola TC18 strain and cloned into Escherichia coli. Based on pTC18, several shuttle vectors, containing Prevotella/Bacteroides tetQ selection marker and E. coli vector püC19 inserted at two different positions in pTC18 were constructed. The shuttle vectors, protected with HaelII methylase against the P. ruminicola 23 restriction were electroporated into P. ruminicola. Despite numerous attempts a tetracycline resistant recombinant strain 23 was not obtained. The possible causes for electroporation failure are discussed.
Key words: microbiology / anaerobic bacteria / Prevotella ruminicola / shuttle vector / rumen
Osamitev plazmida pTC18 seva Prevotella ruminicola TC18 in njegova uporaba v razvoju prenosljivih vektorjev Escherichia coli-P. ruminicola
V vampnem sevu Prevotella ruminicola TC18 smo odkrili 3 kilobazne pare dolgo plazmidno DNA, jo poimenovali pTC18 in klonirali v Escherichia coli. Na njeni osnovi smo razvili več različic prenosljivega plazmida, ki je poleg pTC18 vseboval še selekcijski marker tetQ iz sevov rodu Bacteroides in plazmidni vektor E. coli pUC19. Prenosljive vektorje smo s HaelII meti-lazo zaščitili proti restrikciji v P. ruminicola 23 in jih nato poskusili vnesti v P. ruminicola 23 z elektrotransformacijo. Kljub mnogim poskusom nismo uspeli pridobiti proti tetraciklinu odpornih sevov P. ruminicola 23.
Ključne besede: mikrobiologija / anaerobne bakterije / Prevotella ruminicola / prenosljivi vektor / vamp
1 INTRODUCTION
Prevotella ruminicola is thought to be the most numerous among the strictly anaerobic gram negative rumen bacteria from the genus Prevotella which apparently play important roles in the rumen ecosystem (Tajima et al., 2001; Miyazaki et al., 2003). The genome of the P. ruminicola type strain 23 is currently being sequenced at former TIGR, now J. Craig Venter Institute (http://www. jcvi.org/rumenomics/). However, even the most basic genetic tools such as gene introduction system, which would enable verification of ideas that may originate from the genome data analysis, are undeveloped for this bacterial species. It was shown previously (Purdy et al., 2002) in Clostridium difficile that plasmids, native to spe-
cies to be genetically manipulated are needed and restriction barriers must be characterized and circumvented in order to develop a successful gene transfer system. To construct shuttle vectors for P. ruminicola, native P. ruminicola plasmids are therefore needed. Plasmids, however are surprisingly scarce in this bacterial genus (Peterka et al., 2003). One of the few reported P. ruminicola plasmids was found in P. ruminicola strain TC18 but was not characterized nor exploited as a shuttle vector (Avguštin, 1992). Recently, the type II restriction-modification system of P. ruminicola 23 was described as well as a procedure using HaelII methylase to protect DNA against it was developed (Accetto et al., 2005).
1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, Ph.D., M.Sc., e-mail: tomaz.accetto@bfro.uni-lj.si
2	MATERIAL AND METHODS
2.1 STRAIN, PLASMID, MEDIUM AND GROWTH
P. ruminicola TC18 (van Gylswyk, 1990) was grown anaerobically in M2 medium (Hobson, 1969) according to the Bryant's modification of the Hungate technique (Bryant, 1972). Source of tetQ alele was E. coli-Bacter-oides shuttle plasmid pRH3 (Daniel et al., 1995).
The plasmid Dna was extracted using standard alkaline lysis. Cleavage with restriction endonucleases, ligation and transformation of Escherichia coli were all done using standard molecular biology techniques (Sam-brook, 2001). The Dna was protected against the P. ruminicola 23 restriction using HaeIII methylase (Neb, uSA) according to manufacturers instructions in reactions which contained S-adenosyl methionine as the methyl donor. The protected plasmid DNA was electro-porated into P ruminicola TC18 as described previously (Accetto et al., 2005). Briefly: growth of P. ruminicola TC18 culture was stopped during exponential growth at OD600 = 0.5 by chilling on ice. The cells were then washed three times in anaerobic ice-cold 10% glycerol, electro-porated at 12.5 kV/cm, resuspended in fresh M2 medium and left at 37 °C for an hour. Subsequently, the 0.1 ml portions of cells were transferred on tetracycline containing M2 agar plates in an anaerobic chamber.
3	RESULTS AND DISCUSSION
plasmid DNA was isolated from P. ruminicola TC18 (Fig. 1A). Restriction enzymes HindIII, BamHI, KpnI in
XhoI all convert plasmid DNA into a linear, approximately 3100 base pairs long DNA. The plasmid was named pTC18 and its restriction map is presented in fig. 1B.
HindIII cleaved pTC18 was ligated into multiple cloning site of puC19 and transformed into E. coli TOp10 (Invitrogen, uSA). The resulting construct was cleaved using SstI and ligated to tetQ allele. The latter was obtained by cleavage of pRH3 with SstI and subsequent isolation of 2.6 kilobase pair fragment from the agarose gel. The ligation products were transformed into E. coli TOp10 and restriction analysis of plasmid DNA was performed on several recombinant strains to obtain strains harbouring both possible tetQ orientations (fig. 2)
Since it is possible that HindIII site lies within the pTC18 replication region and thus cloning into this site would most likely inactivate replication in Prevotella hosts, we have also constructed shuttle vectors using the pTC18 KpnI site. The procedures were essentially the same as above yielding constructs presented in figure 3.
All four shuttle vector constructs were subsequently protected against the P. ruminicola 23 restriction enzyme Pru2I using HaeIII methylase and electroporated into P. ruminicola 23 cells. Despite numerous attempts we were unable to obtain a tetracycline resistant P. ruminicola 23 strain harbouring the shuttle vector. The electroporation parameters i.e. DNA concentration, electrocompetent cells density and electroporation time constant were essentially the same as in the previously described successful electroporation of plasmid pRH3 into P. bryantii TC1-1 strain (Accetto et al., 2005). Several explanations for the failure of electroporation are possible: (i) both, HindIII and KpnI site are placed within the region essential for pTC18 replication (ii) P. ruminicola 23 harbours
A 1 2	B
KpnI (1)
SaniHI i:mü)
Figure 1: A: Plasmid DNA isolated from P. ruminicola TC18, agarose DNA electrophoresis. 1: marker generuler 1kb dna ladder (Fer-mentas); 2: plasmid DNA isolated from P. ruminicola TC18. B: Restriction map of pTC18.
Slika 1: A: Plazmidna DNA iz P. ruminicola TC18, agarozna DNA elektroforeza. 1: velikostni standard generuler 1kb dna lestvica (Fermentas); 2: plazmidna DNA, osamljena iz P. ruminicola TC18. B: Restrikcijska mapa pTC18.
Figure 2: Restriction maps of shuttle vectors based on pTC18 cleaved with HindlII and with different orientations of tetQ gene. Slika 2: Restrikcijska mapa prenosljivih vektorjev osnovanih na pTC18 cepljenim s HindIII z različnima usmeritvama tetQ.
another, non type II restriction system (iii) P. ruminicola 23 contains a cryptic plasmid that cannot be isolated by ordinary means or its relicts, but in both cases they belong to the same incompatibility group as pTC18 does and (iv) tetQ gene is lethal to or does not function in P. ruminicola 23.
4 CONCLUSIONS
The novel Prevotella plasmid pTC18 based shuttle vectors were unable to transform P. ruminicola 23. Several strategies to overcome this may be envisaged: transformation of other P. ruminicola strains preceded
by protection of transforming Dna using cell free extract of strains to be transformed (Accetto et al., 2005); the tetQ antibiotic resistance gene can be exchanged with cfXA2 cephalosporinase resistance gene, known to reside in several oral Prevotella isolates (Giraud-Morin et al., 2003) and finally, the other two unique restriction sites BamHI and XhoI can be exploited as cloning sites for antibiotic resistance gene and E. coli replicon in order to evade the pTC18 replication region supposedly inactivated by cloning into HindIII and KpnI sites.
Figure 3: Restriction map of shuttle vectors based on pTC18 cleaved with KpnI and with different orientation of tetQ gene. Slika 3: Shema prenosljivih vektorjev osnovanih na pTC18 cepljenim s KpnI z različnima usmeritvama tetQ.
5 REFERENCES
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