Inverse PCR to localise insertion sites of integrating mobile elements: location of the insertion site of the bacterial genomic island R391.
Inverse PCR to localise insertion sites of integrating mobile elements: location of the insertion site of the bacterial genomic island R391.
J. Tony Pembroke and Barry M. McGrath, Molecular Biochemistry Laboratory, University of Limerick, Ireland.
Abstract
We have utilised an inverse PCR technique to localise the insertion site of the mobile bacterial genomic island R391 and other members of this group. Based on the 89-kb sequence of this integrating element, PCR primers were designed in the forward and reverse direction to sequences at the ends of the element but facing away from each other. Chromosomal DNA containing the element was restricted by a number of four base pair cutting restriction enzymes and each digest separately ligated. The idea being that fragments containing the junction point between the element and chromosome of the host i.e. the insertion point, would circularise and now the primer pair would point towards each other. Standard PCR reactions were then used to allow amplification of the specific junction fragments. This amplicon was then sequenced. In the case of R391 and its relatives R997, pMERPH, R705, R992 and SXT the sequence indicated that integration occurred into the prfC gene of its enteric host. This technique can be utilised to locate the insertion site of any genomic island or phage for which sequence information is available.
Introduction
Genomic analysis of large numbers of bacterial genomes has revealed that there appears to be a ‘core’ genome of essential bacterial genes and a ‘flexible’ genome of acquired DNA, which was probably acquired by horizontal gene transfer. Such islands include pathogenicity islands, symbiosis islands, conjugative transposons, fitness islands and integrating antibiotic cassettes (Pembroke et al 2002). Many such islands integrate into tRNA genes, possibly because they are redundant and widely distributed amongst bacteria allowing broad host range spread upon horizontal gene transfer. Although tRNA genes are common insertion sites there are other sites, such as the gal-bio site for lambda integration, and locating the insertion site of many elements can be problematic. We have been analysing the molecular biology of one type of genomic fitness island, R391 and its relatives R997, pMERPH, R705 and R392. These elements integrate into the bacterial chromosome; encode antibiotic resistance and a number of UV inducible functions, which confer fitness functions in certain bacterial hosts (Murphy and Pembroke1999; Pembroke et al 2002). The element forms a non replicative circular intermediate upon transfer (McGrath and Pembroke 2005) and this has allowed its isolation and sequencing (Boltner et al 2002). We have also confirmed site-specific integration via pulsed Field Gel electrophoresis (Pembroke and McGrath 2005), as it is important to determine whether there are single or multiple sites of integration of such elements. We have adapted a PCR strategy to locate the insertion site of R391 and other related elements. Recently there have been a number of new reports of R391-like elements emerging in human epidemic outbreaks of cholera worldwide and in fish pathogens (Dalsgaard et al 2001; Phantouamath et al 2001;Iwanaga et al 2004; Ehara et al 2004; Amhed et al 2005; JuiZ-Rio et al 2005) with these elements encoding antibiotic resistance and possibly pathogenicity functions.
Conventional PCR requires that at least part, or all, of the DNA sequence in the region to be amplified is known (Saiki et al., 1988). The fact that the sequences of the R391 ends that insert into the chromosome were unknown at this point posed a problem in using a standard PCR approach to amplify across the R391-E. coli junctions. An inverse PCR (IPCR) strategy, which facilitates identification of unknown DNA flanking regions of known DNA (Ochman et al., 1988; Silver, 1991; Triglia et al., 1988), was adopted to amplify the junction fragments. Based on the sequence at the ends of the R391 element (Boltner et al 2002) two primers were initially designed to direct replication away from each other. After digesting R391-containing E. coli chromosomal DNA with restriction enzymes that cut outside the candidate site(s) and ligating the restriction fragments generated, the IPCR primers end up facing each other, thereby allowing amplification of the R391-E.coli chromosome junction fragments.
Sequence analysis of these IPCR amplicons has allowed location and analysis of the junction fragments. Figure 1, illustrates an alignment of the att L and att R sites for a number of R391 and related elements determined by the IPCR technique outlined
Element attL core sequence attR core sequence
R391 ATT ATT TCT CAC CCT GA ATC ATC TCT CAC CCG GA
R392 ATT ATT TCT CAC CCT GA ATC ATC TCT CAC CCG GA
R705 ATT ATT TCC CAC CCT GA ATC ATC TCT CAC CCG GA
R997 ATT ATT TCC CAC CCT GA ATC ATC TCC CAC CCG GA
pMERPH ATT ATT TCT CAC CCT GA ATC ATC TCT CAC CCG GA
Figure 1. Comparison of the proposed core attL and attR sequences of R391, R392, R705, R997 and pMERPH. Underlined nucleotides (at position 9 in each sequence) indicate where these sequences differ.
Conclusion
IPCR has proven useful in determining the genomic island/ chromosomal junctions and integration sites of R391 and a number of related elements.
In principle IPCR can be utilised as a tool to determine the integration junctions not just of genomic islands but almost any integrating DNA in any organism.
References
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