Multiple controls regulate the expression of mobE, an HNH homing endonuclease gene embedded within a ribonucleotide reductase gene of phage Aeh1

Abstract

Mobile genetic elements have the potential to influence the expression of genes surrounding their insertion site upon invasion of a genome. Here, we examine the transcriptional organization of a ribonucleotide reductase operon (nrd) that has been invaded by an HNH family homing endonuclease, mobE. In Aeromonas hydrophila phage Aeh1, mobE has inserted into the large-subunit gene (nrdA) of aerobic ribonucleotide reductase (RNR), splitting it into two smaller genes, nrdA-a and nrdA-b. This gene organization differs from that in phages T4, T6, RB2, RB3, RB15, and LZ7, where mobE is inserted in the nrdA-nrdB intergenic region. We present evidence that the expression of Aeh1 mobE is regulated by transcriptional, posttranscriptional, and translational controls. An Aeh1-specific late promoter drives expression of mobE, but strikingly the mobE transcript is processed internally at an RNase E-like site. We also identified a putative stem-loop structure upstream of mobE that sequesters the mobE ribosome binding site, presumably acting to down regulate MobE translation. Moreover, our transcriptional analyses indicate that the surrounding nrd genes of phage Aeh1 are expressed by a different strategy than are the corresponding phage T4 genes and that transcriptional readthrough is the only mechanism by which the promoterless Aeh1 nrdB gene is expressed. We suggest that the occurrence of multiple layers of control to limit the expression of mobE to late in the Aeh1 infection cycle is an adaptation of Aeh1 to reduce any effects on expression of the surrounding nrd genes early in phage infection when RNR function is critical.

FIG. 1.

Genomic organization and regulatory elements of the Aeh1 nrd genomic region. (A) Schematic of the Aeh1 nrd genomic region, based on the complete genome sequence (44). Genes are indicated by rectangles with arrowheads indicating direction of transcription, with gene names in italics. Positions of primers used for primer extension mapping of transcripts are indicated below the nrdA-a and mobE genes. Predicted regulatory elements are indicated. (B) Consensus sequences for the phage Aeh1 early and late promoters displayed in logos format (53). (C) Putative secondary structures of predicted Rho-independent transcriptional terminators downstream of the genes 50, nrdA-b, and nrdB, respectively. Stop codons and poly(U) tracts are underlined and labeled, with the stability of each helix indicated in kcal/mol as predicted by the Mfold server (64). (D) Structure of a putative regulatory stem-loop upstream of mobE. A box indicates the position of the late promoter upstream of mobE, with a vertical line indicating the mobE RBS.

FIG. 2.

Mapping of transcript initiation sites upstream of the nrdA-a gene. (A) Primer extension mapping of the transcription start site using primer DE-110 and RNA isolated 20 min post-Aeh1 infection. An aliquot of the primer extension reaction mixture was electrophoresed alongside a sequencing ladder of the nrdA-a upstream region. The initiating nucleotide is identified by an asterisk. (B) (Top) Primer extension analysis of transcription initiation at the nrdA-a promoter at various times post-Aeh1 infection. (Bottom) Primer extension analysis of RNA isolated after rifampin treatment of Aeh1-infected cells. Rifampin was added 5 min post-Aeh1 infection (indicated by a plus sign). The sequence of the nrdA-a upstream region is shown below with positions of the −35 and −10 boxes of the early promoter and transcriptional start site indicated relative to the nrdA-a ATG codon. The RBS is underlined. (C) Partial sequences of four clones from 5′-RLM-RACE analysis of nrdA-a initiated transcripts aligned with the genomic sequence (gDNA).

FIG. 3.

Mapping of transcript initiation sites upstream of the mobE gene. (A) Representative primer extension mapping of the transcription start site using primer DE-120, electrophoresed alongside a sequencing ladder generated with the same primer. The sequence of the mobE upstream region and the structure of the predicted stem loop are indicated to the right of the gel. Potential initiating nucleotides (T-23 and G-22) are indicated by arrows, while the G-10 and T-9 sites are indicated by asterisks. (B) Experimental outline to distinguish initiated from processed transcripts using TAP as elaborated in the text. P, 5′ phosphate; open rectangle, RNA adaptor oligonucleotide. (C) Agarose gel of 5′-RLM-RACE reactions using RNA treated with (+) or without (−) TAP. Aliquots of the mixture from the final nested PCR step were electrophoresed alongside a pBR322/AluI ladder. (D) Sequences of five clones corresponding to each of the 5′-RLM-RACE products in panel B. Only partial sequences of the clones are shown. The sequences are aligned with the genomic sequence (gDNA) of the mobE coding region. The late promoter (P_L), start codon, and putative RNase E sites are indicated. (E) Summary of transcript mapping and 5′-RLM-RACE data indicating that late-initiated transcripts would not include sufficient sequence to form a regulatory hairpin that sequesters the mobE RBS.

FIG. 4.

Northern blot analyses of the Aeh1 nrd operon. (A) Northern blot analysis of RNA isolated immediately before Aeh1 infection and at various times post-Aeh1 infection using a probe directed against the full-length nrdA-a gene. The sizes of the hybridizing bands are indicated. (B and C) Northern blot analyses as in panel A but using probes directed against the mobE and nrdB genes, respectively.

FIG. 5.

Transcription of the bacteriophage Aeh1 nrd operon as determined by RT-PCR analyses. Positions of primer pairs used in RT-PCR are indicated on a schematic of the nrd operon, along with the sizes of the expected amplicons. (A to G) Individual agarose gels corresponding to primer pairs as shown above. Lanes for each gel: 1, amplification with genomic Aeh1 DNA (gDNA); 2, RT-PCR performed without prior reverse transcriptase reaction; 3, RT-PCR performed with RNA isolated pre-Aeh1 infection of A. hydrophila; 4, RT-PCR performed with RNA isolated 30 min post-Aeh1 infection; 5, RT-PCR performed without RNA. Relevant sizes of the DNA standard are indicated alongside each gel.

FIG. 6.

Transcription termination and readthrough at Rho-independent terminators downstream of the nrdA-b and nrdB genes. (A) Schematic of the nrdA-b and nrdB genes with the positions of probes used for RPAs. The sizes of protected fragments corresponding to termination and readthrough are indicated. (B and C) 3′-RLM-RACE sequence results of transcripts terminating at the nrdA-b and nrdB terminators, respectively. Partial sequences of cloned 3′-RLM-RACE products are aligned with the genomic sequences (gDNA) corresponding to the 3′ regions of the nrdA-b and nrdB genes, respectively. The stem-loop structure of each terminator is indicated on the genomic sequence. (D and E) RPAs showing the ratio of readthrough transcription to termination at various times at the nrdA-b and nrdB terminators, respectively. For each protection assay, aliquots of the reaction mixtures were electrophoresed alongside a labeled 100-bp ladder (M). The percentage of product corresponding to termination or readthrough is indicated below each gel. −, probe only; +, probe digested with RNase.

FIG. 7.

Summary of transcriptional regulation in the nrd genomic region of phages Aeh1 and T4, labeled as in Fig. 1. Transcripts are indicated by lines, with approximate sizes of each transcript indicated to the right. Dashed lines indicate transcripts that initiate upstream of the nrdA gene of Aeh1 and T4, while the parentheses around the Aeh1 transcripts indicate that the initiation point has not been mapped. Transcripts for the phage T4 region are based on published material as described in the text (56, 59, 60).