Bacteriophage T7, considered the “model phage” of the
Autographiviridae
family, is marked by a strictly lytic life cycle and conserved genome organization. Recently, novel phages within this clade have emerged which display characteristics associated with a temperate life cycle.
Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long 800 nm tail. We found that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular and viral two-barrel RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We determined the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab claw-like architecture similar to previously reported two-barrel RNAPs. The virion RNAP adopts a unique flat structure without a clamp, which likely reflects the requirement for its extrusion through the narrow channel in the phage tail for delivery into the cell. Structure and sequence comparisons of the P23-45 RNAPs with other phage and cellular RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate remarkable adaptability of two-barrel RNAPs that can be attained within a single virus species.
Efficient transcriptional
terminators are essential for
the performance
of genetic circuitry in microbial SynBio hosts. In recent years, several
libraries of characterized strong terminators have become available
for model organisms such as Escherichia coli. Conversely, terminator libraries for nonmodel species remain scarce,
and individual terminators are often ported over from model systems,
leading to unpredictable performance in their new hosts. In this work,
we mined the genomes of Pseudomonas infecting phages LUZ7 and LUZ100 for transcriptional terminators
utilizing the full-length RNA sequencing technique “ONT-cappable-seq”
and validated these terminators in three Gram-negative hosts using
a terminator trap assay. Based on these results, we present nine terminators
for E. coli, Pseudomonas
putida, and Pseudomonas aeruginosa, which outperform current reference terminators. Among these, terminator
LUZ7 T50 displays potent bidirectional activity. These data further
support that bacteriophages, as evolutionary-adapted natural predators
of the targeted bacteria, provide a valuable source of microbial SynBio
parts.
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