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, 187 (23), 7945-54

Enterobacterial Repetitive Intergenic Consensus Sequence Repeats in Yersiniae: Genomic Organization and Functional Properties

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Enterobacterial Repetitive Intergenic Consensus Sequence Repeats in Yersiniae: Genomic Organization and Functional Properties

Eliana De Gregorio et al. J Bacteriol.

Abstract

Genome-wide analyses carried out in silico revealed that the DNA repeats called enterobacterial repetitive intergenic consensus sequences (ERICs), which are present in several Enterobacteriaceae, are overrepresented in yersiniae. From the alignment of DNA regions from the wholly sequenced Yersinia enterocolitica 8081 and Yersinia pestis CO92 strains, we could establish that ERICs are miniature mobile elements whose insertion leads to duplication of the dinucleotide TA. ERICs feature long terminal inverted repeats (TIRs) and can fold as RNA into hairpin structures. The proximity to coding regions suggests that most Y. enterocolitica ERICs are cotranscribed with flanking genes. Elements which either overlap or are located next to stop codons are preferentially inserted in the same (or B) orientation. In contrast, ERICs located far apart from open reading frames are inserted in the opposite (or A) orientation. The expression of genes cotranscribed with A- and B-oriented ERICs has been monitored in vivo. In mRNAs spanning B-oriented ERICs, upstream gene transcripts accumulated at lower levels than downstream gene transcripts. This difference was abolished by treating cells with chloramphenicol. We hypothesize that folding of B-oriented elements is impeded by translating ribosomes. Consequently, upstream RNA degradation is triggered by the unmasking of a site for the RNase E located in the right-hand TIR of ERIC. A-oriented ERICs may act in contrast as upstream RNA stabilizers or may have other functions. The hypothesis that ERICs act as regulatory RNA elements is supported by analyses carried out in Yersinia strains which either lack ERIC sequences or carry alternatively oriented ERICs at specific loci.

Figures

FIG. 1.
FIG. 1.
ERIC elements in yersiniae. (A) ERIC-sized classes in Y. enterocolitica 8081 and Y. pestis CO92 strains. The number of elements carrying both TIRs found and their sizes in base pairs are indicated. (B) Structural organization of ERICs. Boxed arrows mark the TIRs. Triangles mark type 1-to-type 3 insertions interrupting ERIC sequences. (C) The consensus sequence of the 127-bp unit-length ERICs is shown in the A orientation. TIR residues are in capital letters. Underlined residues mark sequences conserved in the internally rearranged 70-bp-long ERICs. The integration sites of type 1-to-type 3 insertions are denoted by asterisks. (D) The consensus sequences of the three types of intervening sequences found to interrupt ERICs are shown.
FIG. 2.
FIG. 2.
Filled and empty ERIC sites. Homologous DNA regions from the Y. enterocolitica 8081 (Ye) and Y. pestis CO92 (Yp) strains are aligned. Numbers refer to genome residues; dashes denote sequence identities. The duplication of the TA target site at ERIC termini is highlighted.
FIG. 3.
FIG. 3.
Primer extension analyses of ERIC-positive transcripts. Primers that had been 32P labeled at the 5′ end and were complementary to lpdA, uncE, cheW, and trpB transcripts were hybridized to total RNA (5 μg) derived from the Y. enterocolitica Ye161 strain. Annealed primer moieties were extended in the presence of nucleoside triphosphates by avian myeloblastosis virus reverse transcriptase. Reaction products were electrophoresed on 6% polyacrylamide-8 M urea gels. Major reaction products labeled “a” to “l” are marked by arrows. Numbers to the left of each autoradiogram refer to the size in nucleotides of coelectrophoresed DNA molecular size markers. In the diagrams at the bottom are sketched the organizations of the ERIC-positive regions analyzed. The direction of transcription of the genes analyzed is indicated by dotted arrows. Primers are denoted by arrows; lines labeled “a” to “l” denote the extended products. Numbers indicate the distances in base pairs separating ERICs from either the stop or the start codons of neighboring ORFs.
FIG. 4.
FIG. 4.
Asymmetry in the orientations of ERICs. The distances in base pairs separating B-oriented and A-oriented ERICs from flanking upstream ORFs in the Y. enterocolitica 8081 strain are indicated.
FIG. 5.
FIG. 5.
RT-PCR analyses of ERIC-positive transcripts. Total RNA (200 nanograms) derived from the Ye161 strain had been reverse transcribed by using a mixture of random hexamers as primers. The cDNA obtained had been amplified by PCR with cistron-specific oligomers. One oligonucleotide of each pair of primers was 32P end labeled to allow amplimer detection by autoradiography. Reaction products were run on 6% polyacrylamide-8 M urea gels. (A) Transcripts spanning ERIC sequences and cheA-cheW (lane 1), cheB-cheY (lane 2), manX-manY (lane 3), panB-panC (lane 4), trpC-trpB (lane 5), and pstS-pstC (lane 6) genes were detected by using external primers 1 and 4 under standard PCR cycling conditions (20 to 22 cycles). Amplimers were detected only when RNA samples were incubated with reverse transcriptase (+ lanes) prior to PCR. (B) Total cDNA from the Ye161 strain had been amplified by using pairs of ORF-specific primers for a limited number of PCR cycles (6 to 12). Amplimers were quantitated by phosphorimaging. In the example reported, amplimers 1 and 2 correspond to the cheA and cheW genes, respectively (C) The listed genes flanking ERIC repeats have been analyzed as described above. Distances in base pairs separating ERIC termini from stop and start codons of flanking ORFs are indicated. The orientation of the ERIC (A or B) is given in parenthesis. The number of transcripts corresponding to downstream (dw) and upstream (up) genes for each pair is expressed as a ratio. RT-PCR analyses were routinely repeated three to four times on two independent RNA preparations. Standard deviations are indicated. For each ORF analyzed (with the YE number assigned by the Sanger Centre shown in parentheses), the hypothesized function, system, and/or product(s) are as follows: for cheA (YE2577), chemotaxis protein CheA; for cheW (YE2576), chemotaxis protein CheW; for trpC (YE2212), tryptophan biosynthesis bifunctional protein; for trpB (YE2213), tryptophan synthase subunit B; for phoT (YE4198), high-affinity P-specific transport and cytoplasmic ATP-binding protein; for phoU (YE4196), P uptake, high-affinity P-specific transport system, and regulatory gene; for cheB (YE2571), glutamate methylesterase; for cheY (YE2570), chemotaxis protein CheY; for glgC (YE4011), glucose-1-phosphate adenylyltransferase; for glgA (YE4010), glycogen synthase; for manX (YE1777), mannose phosphotransferase system and EIIAB component; for manY (YE1776), mannose phosphotransferase system and EIIC component; for panB (YE0720), ketopantoate hydroxymethyltransferase; for panC (YE0719), pantoate-beta-alamine ligase; for pstS (YE4201), phosphate-binding periplasmic protein; and for pstC (YE4200), phosphate transport system permease.
FIG. 6.
FIG. 6.
RNase protection of ERIC-positive transcripts. Uniformly 32P-labeled antisense RNA probes, complementary to the coding regions of the Y. enterocolitica cheB-cheY and panB-panC genes, were transcribed in vitro by the T7 RNA polymerase. In the diagram, RNA probes are sketched (not to scale) below the gene depictions. Thicker segments mark complementarity to mRNA. Probes were hybridized to 20 μg of total RNA from Ye161 cells untreated or exposed to rifampin (final concentration, 200 μg/ml) for 12 min before cell harvesting. RNase T1-resistant RNA hybrids were electrophoresed on 6% polyacrylamide-8 M urea gels. Reaction products corresponding to cheY, cheB, panC, and panB RNAs are marked by arrows. (A) Analysis of cheB-cheY RNAs. Lanes: unreacted input probes (1, cheY; 5, cheB); probes hybridized separately to total RNA from Ye161 cells untreated (2, cheY; 6, cheB) or exposed for 12 min to rifampin (3, cheY; 7, cheB) or hybridized to E. coli tRNA (4, cheY; 8, cheB). Probes were hybridized together to total RNA from Ye161 cells untreated (9) or exposed for 12 min to rifampin (10) or hybridized to E. coli tRNA (11). (B) Analysis of panB-panC RNAs. Lanes: unreacted input probes (12, panC; 16, panB); probes hybridized separately to total RNA from Ye161 cells untreated (14, panC; 18, panB) or exposed for 12 min to rifampin (15, panC; 19, panB) or hybridized to E. coli tRNA (13, panC; 17, panB). Probes were hybridized together to total RNA from Ye161 cells untreated (20) or exposed for 12 min to rifampin (21) or hybridized to E. coli tRNA (22).
FIG. 7.
FIG. 7.
Comparison of loci carrying or missing ERIC sequences in different Yersinia strains. ERIC elements are depicted as gray boxes, and numbers within refer to element sizes. Numbers above boxes signal the distances in base pairs separating ERIC from flanking ORFs. Total RNAs derived from Y. enterocolitica strains Ye161 and Ye25 and from Y. kristensenii strain YkSS47 were analyzed by RT-PCR as described for Fig. 5. At the empty genomic sites identified in the genome of the YkSS47 strain, ERIC sequences are replaced by the dinucleotide TA.
FIG. 8.
FIG. 8.
(A) Predicted RNA foldings and relative calculated free energies at 37°C of unit-length ERIC consensus sequences inserted in A and B orientations. Non-Watson-Crick base pairings are highlighted by dots. The hypothesized cleavage site for RNase E, present in B-oriented ERICs, is boxed. (B) Translation-dependent processing of ERIC-positive RNAs. Total RNA derived from exponentially growing Ye161 cells untreated (−) or exposed for 30 min to chloramphenicol (+) (final concentration, 50 μg/ml) was analyzed as described for Fig. 5. (C) Ribosomes interfere with folding of ERIC-positive RNA. In mRNA-spanning B-oriented elements that are inserted close to the translational stop codon, the translating ribosome covers most of the ERIC left-hand TIR, unmasking the RNase E site (sketched as a triangle) located in the ERIC right-hand TIR.

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