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Klinische Forschergruppe, OE 6710, Medizinische Hochschule Hannover, D-30625 Hannover, Germany
Correspondence
Jens Klockgether
klockgether.jens{at}mh-hannover.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 1997) that harbour the most abundant genomic islands in the P. aeruginosa population. The related pKLC102-type and PAGI-2-type islands are present in 
70 and 40 % of P. aeruginosa strains, respectively (Klockgether et al., 2007). pKLC102-type islands (He et al., 2004; Klockgether et al., 2004) integrate into one of the two copies of a tRNALys gene at positions PA0976.1 and PA4541.1 of the PAO1 reference genome (Stover et al., 2000), whereas PAGI-2 type islands preferentially target one of the two tRNAGly genes at position PA2819.1-2 (Larbig et al., 2002). pKLC102-type and PAGI-2-type islands belong to an ancient family of genomic islands in proteobacteria and share a syntenic backbone of 36 homologues that include genes related to DNA replication or genetic mobility, or that encode conserved hypothetical proteins of unknown function (Juhas et al., 2007; Mohd-Zain et al., 2004). The islands differ in genetic mobility. PAGI-2-type islands spread across species barriers to other proteobacteria (Gaillard et al., 2006; Müller et al., 2003), but their spontaneous excision rates are well below 10–7 (Klockgether et al., 2007), and the expression of the integrase is under stringent regulation (Sentchilo et al., 2003a, b). pKLC102, however, is the most mobile genomic island known to date (Klockgether et al., 2007). Being a hybrid of phage and plasmid elements, the spontaneous excision rate from the host chromosome is at least 10–1. The copy number of extrachromosomal circular pKLC102 in the cell varies between one and 30 during bacterial growth. Qiu et al. (2006) have proven that the pKLC102-type island PAPI-1 can be transferred by conjugation into other P. aeruginosa strains, where it integrates into one tRNALys gene of the recipient chromosome.
We wanted to know whether the differential mobility of the island types is mirrored by differential utilization of the encoded genes. Taking the completely sequenced islands PAGI-2 and pKLC102 as prototypes, the clone C strains C and SG17M (Römling et al., 1997) were assayed for mRNA expression of the 103 pKLC102 and 111 PAGI-2 ORFs. PAGI-2 ORFs were not transcribed or were weakly transcribed under standard growth conditions, whereas the mRNA transcripts of the mobile pKLC102 made up at least 10 % of the RNA of the bacterial cell. For selected ORFs, the transcript level was determined in detail by semiquantitative RT-PCR.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). PAO DSM 1707 was used as the P. aeruginosa reference strain. Bacteria were grown at 37 °C in either Luria–Bertani (LB) broth or modified Vogel–Bonner (VB) medium (7.6 mM sodium citrate, 28 mM NaNH4HPO4, 37 mM K2HPO4, 3.3 mM MgSO4, 213.4 mM potassium-(D)-gluconate; pH 7.2). Cells were harvested in the exponential phase at OD578 1.0 and in the stationary phase at OD578 3–4.
PAGI-2 and pKLC102 macroarrays.
The macroarrays (Klockgether et al., 2007) consisted of Hybond-N+ membranes (Amersham), on which 208–805 bp PCR-amplified sequences of pKLC102 or PAGI-2 had been spotted in a 96 (8x12) dot format. For the PAGI macroarrays, 91 PCR products were distributed onto the membrane representing 94 of the 111 predicted ORFs from PAGI-2. ORF C47 was represented by two different products (‘C47c’ and ‘C47d’). C54 and C55, C68 and C69, C76 and C77, and C82 and C83 were represented pair-wise by one product each. In the pKLC102 macroarray, 86 PCR products represented 85 ORFs of pKLC102. Three PCR products represented the 2.9 kb ORF CP94 (‘CP94a’, ‘CP94b’ and ‘CP94c’) and two products represented ORF CP103 (‘CP103a’ and ‘CP103b’). Three PCR products spanned two ORFs each (CP47–CP48, CP52–CP53, and CP73–CP74). Control PCR products were spotted in the lower-left corners. In the case of the PAGI-2 array, the five dots contained partial sequences of (from top to bottom) the P. aeruginosa genes gltA, fliC (type A) and fliC (type B) (positive controls), and of an intergenic sequence of Pseudomonas putida KT2440 and of the human ob gene (negative controls). In addition to these five control dots, the pKLC102 macroarray contained, in the second lane from the left, the five controls, ori1' of pKLC102, PA0977 and PA0981 of P. aeruginosa PAO1, and two strain-specific ORFs of P. aeruginosa TBCF10839.
Copies of the macroarrays were produced in parallel from the same set of PCR products (pooled from four reactions each). Aliquots (50 µl) of these pooled products, 15 µl 3 M NaOH and 85 µl Tris-EDTA buffer were denatured at 65 °C for 30 min and chilled on ice. After adding 100 µl 3 M ammonium acetate, aliquots of 100 µl per dot were transferred to the membranes by a Minifold dot-vacuum-blot apparatus (Schleicher & Schuell), thus generating array copies with an identical amount of PCR product of the same DNA quality at comparable positions. After spotting, the DNA was cross-linked to the membranes by 1 min exposure to UV light. Control stripes for the calibration of expression levels contained two controls also used for the macroarrays (gltA, ob) and four genes of the P. aeruginosa core genome (Stover et al., 2000) with strongly different mRNA transcript expression levels [gltA, rpoN, rpsL and rrn (two segments of the 16S rDNA sequence)].
RNA isolation and cDNA synthesis.
Bacterial RNA from 1010 P. aeruginosa cells grown in LB broth was isolated by a modified hot phenol method (von Götz et al., 2004). For cDNA synthesis, 10 µg bacterial RNA and 750 ng random primer p(dN6) (Roche) in 30 µl water were incubated at 70 °C for 10 min and at 25 °C for an additional 10 min. After chilling on ice, 12 µl 5x first strand buffer (Invitrogen), 6 µl 0.1 M DTT, 3 µl dNTP-solution (dATP, dTTP, dCTP and dGTP, 10 mM each), 1.5 µl RNase-inhibitor (SUPERaseIn, 20 U µl–1, Ambion) and 7.5 µl reverse transcriptase (Superscript II, 200 U µl–1, Invitrogen) were added and incubated for 10 min at 25 °C, 60 min at 37 °C, 60 min at 42 °C, and finally 10 min at 70 °C. Afterwards, RNA strands were degraded by the addition of 20 µl 1 M NaOH and 30 min incubation at 65 °C. The solution was neutralized with 20 µl 1 M HCl, and the cDNA was purified by using the QIAquick PCR purification kit (Qiagen). The final cDNA concentration was determined spectrophotometrically.
mRNA transcript profiling on macroarrays.
The generated cDNA was cut into fragments of 50–200 bases in length by incubating 12.5 µg cDNA and 6.25 U DNase I (Amersham) in 38 µl OnePhorAll buffer (Amersham) at 37 °C for 10 min, followed by inactivation of DNase I at 98 °C for 10 min. Fragments were labelled at the 3' end with DIG by using the Terminal Transferase kit (Roche).
Labelled cDNA fragments (5 µg) were hybridized onto macroarrays and control stripes. The hybridization (0.5 M sodium phosphate, 1 mM EDTA, 1 % SDS, pH 7.2 for 16 h at 60 °C) was sufficiently stringent so that no cross-hybridization was observed for any of the 36 ORFs that are homologous between the two islands (data not shown). Non-specifically bound probe solution was removed by several washing steps, and hybridization signals were detected by washing the membrane with anti-DIG–alkaline phosphatase (Roche) and CDP Star (Römling et al., 1994). Chemiluminescence signals were detected on X-ray films and quantified by PCBAS, version 2.09f (Raytest Isotopenmeßgeräte).
Semiquantitative RT-PCR kinetics.
The mRNA concentration was determined from the kinetics of amplification of cDNA during PCR by titration for the first reaction cycle with detectable fluorescence of gel-separated PCR product with the ethidium bromide stain (Bremer et al., 1992; Hoof et al., 1991). A PCR product becomes visible by ethidium fluorescence during the late-exponential phase of PCR. Using our PCR instrument (Landgraf), the efficiency, R, of the exponential phase of PCR, N=N0 (1+R)n, was always R=0.78±0.02, within the range of 105–1010 transcript molecules per microgram RNA as the initial cDNA template concentration, if the cDNA PCR product was smaller than 800 bp in size [N, amount of PCR product; N0, initial amount of template (cDNA); n, number of PCR cycles]. The amount of PCR product increased by one order of magnitude within four reaction cycles. The oligonucleotide primer sequences are listed in Table 1.
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RT-PCR kinetics were performed at least four times for each selected mRNA transcript and each experimental condition. The mean and variance of the lowest cycle number with a visible PCR product with the ethidium bromide stain were determined for each set of RT-PCR kinetics by visual inspection of photos of the agarose gels. Cycle numbers were then converted into concentrations in femtomoles mRNA transcript per microgram bacterial RNA with the aid of calibration curves generated by PCR with an MDR1 cDNA standard on the same thermocycler instrument, as described previously by Hoof et al. (1991).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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shows two representative examples of the hybridization of cDNA samples onto pKLC102 and PAGI-2 macroarrays. Hybridization signals of individual pKLC102 and PAGI-2 ORFs did not differ significantly between the four tested growth conditions, each of which was examined at least in duplicate. Absolute signal intensities, however, were strikingly different between pKLC102 and PAGI-2 ORFs (Fig. 1).
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). The majority of the transcribed ORFs belonged to cargo parts of PAGI-2 (most ORFs of C14–C35, C56–C62, C97–C99) that are absent in the related genomic islands PAGI-3 (Larbig et al., 2002), pKLC102 (Klockgether et al., 2004) and PAPI-1 (He et al., 2004). RT-PCR kinetics verified that the transcribed ORFs were each present at less than 1000 copies per microgram of RNA, the exception being C98, with a copy number of about 107 mRNA molecules per microgram of RNA (Table 2). C98 encodes a putative transcriptional regulator that shares extensive homology with a family of response regulators in Pseudomonas species that control detoxification of heavy metal ions (Lee et al., 2001). In summary, all but one of the PAGI-2 ORFs were transcriptionally silent or very weakly expressed under standard in vitro growth conditions.
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In contrast to PAGI-2, ORFs from pKLC102 were expressed significantly under the conditions tested: virtually all ORFs showed moderate to very strong cDNA-reactive hybridization signals (Fig. 1, Table 2). RT-PCR kinetics verified the abundance of pKLC102 mRNA transcripts. Based on an estimate derived from whole-genome sequence data (http://cmr.tigr.org) of 3 pmol RNA molecules (µg RNA)–1, the amount of pKLC102 mRNA transcripts should be at least 10 % of total RNA molecules of the SG17M cell in both exponential and stationary phase. The soj and int genes, which are required for mobilization from and integration into the chromosome (Qiu et al., 2006), as well as the genes defined as the phage and plasmid modules of pKLC102 (Klockgether et al., 2007), belonged to the weakly or moderately expressed genes. Of the 18 most strongly expressed pKLC102 genes (Fig. 1), 11 reside within the contig CP43–CP53. The most abundant mRNA transcripts, CP43 and CP46 (Fig. 1, Table 2), each made up more than 1 % of total RNA of the SG17M cell. Homologues of CP43–CP53 are found in the majority of pKLC102-type islands of P. aeruginosa (Klockgether et al., 2007), with CP43, CP46–CP49 and CP51–53 homologues present in more than 90 % of the islands. Outside the pKLC102/PAGI-2 family of genomic islands, however, no ORF with significant sequence similarity to any of the 11 ORFs could be retrieved from public databases at the time of writing. Hence, bioinformatic tools could not provide any hint as to why this contig is so strongly expressed, at levels close to those of rRNA genes. Functional screens have also been uninformative: the homologous PAPI-1 ORFs of strain PA14 have been shown to be phenotypically inconspicuous in screens for pathogenicity genes in several infection models (He et al., 2004). Of the strongly expressed genes, only CP70 contributes to virulence. CP70 carries the signature of an RND efflux transporter and was the only pKLC102 ORF that was significantly more strongly expressed in mid-exponential than in stationary phase (Table 2).
Seven of the 18 most strongly expressed pKLC102 genes have homologues in PAGI-2. It is interesting to note that none of the homologues was transcribed from the latter at detectable levels. In other words, expression was silent in the stably captured PAGI-2, but extremely high in the mobile and episomally replicating pKLC102, suggesting that the genetic repertoire is utilized in the episomal state and during mobilization, replication or transfer of the genomic island. Differences in copy number, DNA structure and functional needs probably all account for the huge disparity between mRNA expression levels of PAGI-2 and pKLC102 genes under identical environmental conditions. First, the copy number is one for the stably integrated PAGI-2 and up to 30 for the mobile pKLC102. Second, the host chromosome controls the local chromatin structure of its genomic island, PAGI-2, and thus the accessibility of promoters and other regulatory sequences. The circular pKLC102, on the other hand, is largely autonomous in its control of topology and promoter accessibility. Moreover, cis regulatory elements of promoter activity that flank the integration site can cooperate in the circular state, but are physically separated and thus silenced in the chromosomal genomic island (Sentchilo et al., 2003a, b). Third, the genetic information encoded by the genomic island is not essential for the host and hence can be switched off. The selfish episomal form, however, needs its genes to be permanently transcribed for replication and horizontal transfer.
In summary, the mobile gene island pKLC102 is constitutively highly expressed by its host strain SG17M at levels close to those of the most strongly transcribed genes of the core genome, whereas the stably incorporated PAGI-2 is kept transcriptionally silent for most ORFs and probably becomes actively expressed only under exposure to specific environmental cues, as has been described elsewhere for the PAGI-2-type island clc (Gaillard et al., 2006; Müller et al., 2003; Sentchilo et al., 2003b).
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ACKNOWLEDGEMENTS |
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Edited by: D. W. Ussery
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 24 October 2007;
revised 7 March 2008;
accepted 13 March 2008.
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