Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

Abstract

The P R promoter of mycobacteriophage BPs directs early lytic gene expression and is under the control of the BPs repressor, gp33. Reporter gene fusions showed that P R has modest activity in an extrachromosomal context but has activity that is barely detectable in an integrated context, even in the absence of its repressor. Mutational dissection of P R showed that it uses a canonical ؊10 hexamer recognized by SigA, and mutants with mutations to the sequence 5=-TATAMT had the greatest activities. It does not contain a 5=-TGN-extended ؊10 sequence, although mutants with mutations creating an extended ؊10 sequence had substantially increased promoter activity. Mutations in the ؊35 hexamer also influenced promoter activity but were strongly context dependent, and similar substitutions in the ؊35 hexamer differentially affected promoter activity, depending on the ؊10 and extended ؊10 motifs. This warrants caution in the construction of synthetic promoters or the bioinformatic prediction of promoter activity. Combinations of mutations throughout P R generated a calibrated series of promoters for expression of stably integrated recombinant genes in both Mycobacterium smegmatis and M. tuberculosis, with maximal promoter activity being more than 2-fold that of the strong hsp60 promoter.

Genomics and Proteomics of Mycobacteriophage Patience, an Accidental Tourist in the Mycobacterium Neighborhood

“…Patience has a single tRNA gene, and the tRNA is predicted to be charged with glutamine and has the anticodon 5′-UUG [i.e., tRNA Gln (UUG)]. It is unclear how the Patience genes are transcribed, and there are no strongly predicted SigA-like promoters, similar to other mycobacteriophages such as Giles ( 21 ), even though SigA-like promoters are active in other mycobacteriophages ( 22 – 24 ).…”

Comparative Genomics of Cluster O Mycobacteriophages

“…The prediction of mycobacteriophage promoter locations is complicated because while some are related to mycobacterial SigA promoters [ 14 – 16 ], others appear not to be [ 17 ]. However, all five Cluster O phages contain at least eight strongly predicted SigA-like promoters, two rightwards facing (P R2 —P R3 ) and six facing leftwards (P L1 —P L6 ); Corndog, Dylan, and YungJamal have an additional rightwards-facing promoter (P R1 ) upstream of P R2 .…”

Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

“…Plasmid pLO74 is an integrated vector containing the mycobacteriophage Tweety integration cassette (Pham et al ., ) and expresses mCherry from the P hsp60 promoter (Oldfield and Hatfull, ). Wag31 , accA3 ( MSMEG_1807 , coordinates 1,883,433–1,885,229 complement, with start codon changed from GTG to ATG), accD4 ( MSMEG_6391 , coordinates 6,455,190–6,456,743 complement, with start codon changed from GTG to ATG), AccD5 MSMEG_1813 , coordinates 1,888,497–1,890,125) and cwsA ( MSMEG_0023 , coordinates 44,692–45,102) genes of M. smegmatis mc 2 155 were individually cloned into plasmid pLO74 to replace mCherry gene using NEBuilder HiFi DNA Assembly Cloning Kit (NEB) and are named plasmids pCCK25, pCCK26, pCCK27, pCCK28 and pCCK29 respectively.…”

“…Plasmid pLO74 is an integrated vector containing the mycobacteriophage Tweety integration cassette (Pham et al, 2007) and expresses mCherry from the P hsp60 promoter (Oldfield and Hatfull, 2014). Wag31, accA3 (MSMEG_1807, coordinates 1,883,433-1,885,229 complement, with start codon changed from GTG to ATG), accD4 A.…”