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Journal of Bacteriology, May 2005, p. 3586-3588, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3586-3588.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Bacteriophage Mu Targets the Trinucleotide Sequence CGG

Dipankar Manna,¹ Shuang Deng,¹ Adam M. Breier,^2, and N. Patrick Higgins^1*

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294,¹ Graduate Group in Biophysics, University of California, Berkeley, California 94720²

Received 21 December 2004/ Accepted 31 January 2005

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Target specificity for bacteriophage Mu was studied using a new phage derivative that enabled cloning of Mu-host junctions from phage DNA. Insertions distributed throughout the chromosome showed no orientation bias with respect to transcription or replication polarity. Genes with a high frequency of the triplet CGG were preferred targets.

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Mobile genetic elements constitute a significant part of prokaryotic and eukaryotic genomes (2, 17). These elements can have a profound impact on genomic organization and function (3). Among the phages of gram-negative bacteria, bacteriophage Mu is the most efficient transposon known (4, 14). Previous reports indicate that Mu frequently inserts into highly preferred sites within a target gene (6, 13, 20). Using DNA microarray analysis, we recently demonstrated that Mu exhibits a 1,000-fold bias in Escherichia coli at the whole-gene level (12). Transcription plays a dominant role in causing this bias.

The role played by a local DNA sequence in target bias was unclear. In specific cases the upstream transcriptional control region and sequences overlapping promoters were found to be insertion hot spots (13, 20). To see if promoters are generally preferred target sites, and to assess the contribution of DNA sequence in target choice, a library of 100 Mu-host DNA junctions was cloned and sequenced.

Plaque-forming phage with rare cutting restriction sites. MuNXKan was created from Mucts⁶² (9; GenBank accession no. AF083977) by replacing 784 bp of the Mu mom gene with a 1,266-bp kan gene cassette. The mom gene was deleted to allow efficient phage DNA digestion with restriction endonucleases (8). The kan module includes the restriction sites for NotI and XbaI (Fig. 1). The NotI site was introduced with PCR fragments by using pKD4 (5) as the template and the primer pair KanF (TGTAG GCTGG AGCTG CTTCG CGGCC GCGAA GTTCC TATAC TTTCT AGA; homologous bases are underlined, and the NotI site is italicized) and KanR (GTCGC TTGGT CGGTC ATTTC G). The PCR product was reamplified using the primer pair MomF-KanF (CGATC GGTAA TACAG ATCGA TTATG CCCCA ATAAC CACAT GTAGG CTGGA GCTGC TTCG) and MomR-KanR (AGGAA TCTGA TGTAG CGATA CTGAT TAAAT TTGTG TACCG TCGCT TGGTC GGTCA TTTCG; underlined bases are homologous to the mom gene). The module was introduced into a Mu lysogen using lambda red recombineering (5, 21). Escherichia coli strains lysogenic for MuNXKan were resistant to kanamycin at 50 µg/ml and had lytic and lysogenic properties similar to those of the parent phage (data not shown). The predicted structure of MuNXKan was confirmed by DNA sequence analysis (GenBank accession no. AY860420).

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FIG. 1. Schematic representation of MuNXKan. The genetic organization of MuNXKan is shown on the left (A). Marked are genes for the transcriptional repressor Mu c (cts), transposition proteins A and B, cell lysis factors (lys), phage head and tail proteins, and a kanamycin resistance determinant. Positions of the unique NotI and XbaI restriction sites are shown. The attL and attR denote the left and right MuA binding sites, respectively. Thin lines at Mu ends represent covalently attached host-target DNA (not to scale). (B) Restriction digestion profile of MuNXKan. The patterns of uncut (lane 1), NotI-digested (lane 2), and XbaI-digested (lane 3) MuNXKan DNAs are shown. M denotes molecular weight markers. The arrow on the right indicates the Mu right-arm fragment of 3 kbp in size.

MuNXKan phage growth was initiated by thermoinduction of a lysogen of E. coli strain N99 [F^– galK2 galT22 rpsL]. Phage DNA was purified as described previously (12). Mu-host DNA junctions were cloned by selection for Kan resistance. First, phage DNA was made blunt ended with Klenow polymerase. Then, digestion with XbaI liberated fragments with an average size of 3 kbp (Fig. 1B). Gel-purified fragments were cloned into XbaI- and SmaI-digested pUC19. One hundred recombinant plasmids resistant to Amp and Kan (50 µg/ml each) were individually sequenced.

Mu insertions were distributed throughout the chromosome (Fig. 2A). Eighty-five percent of the insertions were within genes. In E. coli, intergenic segments represent 11% of the genome (2); with 100 sequences, 15% intergenic insertion is statistically indistinguishable from 11%. Intragenic insertions showed no orientation bias with respect to the direction of transcription of the target; 53% were oriented from the left end to the right end of Mu (MuL-R) in the direction of transcription (Fig. 2A). Transposon Tn7 shows a strong bias with respect to the direction of replication fork movement (18, 19). Outside of the region between TerA and TerC, where replication forks move in both directions, 92% of all Tn7 insertions are oriented by the direction of fork movement. Earlier analysis of a small number of insertions had suggested that Mu integration occurs in both orientations within each replichore (16). Present data suggest a weak bias at best, as 58% of insertions were oriented MuL-R in the direction of replication (Fig. 2A).

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FIG. 2. (A) Distribution of MuNXKan insertion sites around the chromosome. Transposition target sites were mapped to the bacterial chromosome. Positions of the replication origin (Ori), the terminus (Ter), and the direction of replication are marked on the map. Mu insertions oriented in the direction of replication (attL followed by attR; diamonds) are distinguished from the ones that are oppositely oriented (circles). Insertions that are in the same orientation as transcription are indicated by open symbols, while gray symbols indicate insertions that are oriented opposite to transcription. Black symbols denote intergenic insertions. (B) Mu insertions are distributed along the length of target gene. Distances of Mu insertions from the start codons of target genes were calculated and are expressed as percentages of the target gene length. The number of insertions at a given distance is then plotted as a function of the relative distance of the insertion from the gene start site. ORF, open reading frame.

To determine if Mu insertions were biased towards the ends of genes, the distance of each intragenic insertion was measured from the starting nucleotide. Mu insertion positions were then calculated as a percentage of target gene length. A plot of the distribution of insertions relative to the start codon of each gene showed no bias to either the 5' or 3' end (Fig. 2B).

Role of target sequence in transposition bias. Our set of genomic clones was tested for sequence specificity. To test sequence preferences, alignments were carried out on all 100 target DNA sequences. Studies by Mizuuchi and Mizuuchi (15) had revealed a consensus NYSRN sequence [N(T/C)(G/C)(G/A)N] for the 5-bp duplicated target site, where N is any nucleotide and the Mu right end is positioned at the 5' end of NYSRN. In the set of 100 random insertions, a nucleotide bias within the 5-bp target site was obvious, but no other pattern was apparent. A weight matrix of the distribution of the 4 nucleotides at each of the five positions of the duplicated target site is shown in Fig. 3A. The weight matrix was calculated based on the equation W_i,j = log₂(f_i,j/p_i), where i is (A, T, G, C), j is (1, 2, 3, 4, 5), W_i,j is the weight of the ith base in the jth position, f_i,j is the observed frequency of the ith base in the jth position in target sequences, and p_i is the frequency of the ith base in the genome of E. coli. The bias at the central three positions was stronger than seen in a previous analysis (15). This could be due to the genome-wide target potential with our assay. It is noteworthy that the newly derived consensus NCGGN is a subset of the previously determined NYSRN consensus sequence.

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FIG. 3. Consensus sequence for the Mu transposition target and its correlation with Mu transposition preference. (A) A weight matrix of the 4 nucleotides at each of the positions of the 5-bp Mu target site is shown. A target DNA consensus sequence is shown below the matrix. (B) Relationship between the calculated frequency of CGG trinucleotides and observed TTP of a gene. CGG frequency values of a total of 4,232 genes for which TTP values were known were calculated and portioned into 17 groups. The binned mean log2(TTP) values of the groups are plotted against the average CGG frequencies of the groups, along with the standard error of each mean log2(TTP) value.

Based on the trinucleotide composition of the E. coli genome sequence (GenBank accession no. NC_000913), on average, CGG occurs at a frequency 0.018, or once every 55 bases. At the gene level, the frequency of CGG varied from 0 to 0.1. To evaluate the impact of DNA sequence on target selection, the correlations between the CGG frequency and the measured microarray transposition target preference (TTP) value (12) were compared for every gene. The correlation coefficient of the previously determined consensus YSR as a frequency versus the log₂(TTP) plot was 0.208, whereas CGG frequency versus log₂(TTP) was 0.229. Because transcription exerts a strong effect on the TTP score, we also calculated a three-gene moving average of CGG frequency. Averaging improved the correlation coefficients of both YSR and CGG to values of 0.274 and 0.295, respectively. CGG was a better target site predictor than YSR in all cases. CGG frequency values were partitioned into 17 intervals, which included all groups with at least 10 genes. A plot of gene CGG frequency versus mean log₂(TTP) shows that the consensus sequence modulates transposition frequency over a sixfold range (Fig. 3B).

The reason Mu prefers the target sequence NCGGN is unknown. It could be caused by specific interactions with MuA (1), with Mu's target selector protein MuB (7), or with host cell accessory factors (10, 11).

 
We thank members of N. P. Higgins' laboratory for critical reading of the manuscript.

This work was supported by NSF grant MCB9122048 and NIH grant GM33143. A.M.B. was supported by a Howard Hughes Medical Institute predoctoral fellowship.

 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294.

Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

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Journal of Bacteriology, May 2005, p. 3586-3588, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3586-3588.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Manna, D. Articles by Higgins, N. P. Search for Related Content PubMed PubMed Citation Articles by Manna, D. Articles by Higgins, N. P.