1a) The ∆pnp and pnp* mutants failed to provide any signal upon

1a). The ∆pnp and pnp* mutants failed to provide any signal upon immunoblotting bacterial cell lysates for PNPase, whereas pnp− mutant revealed an expected truncated variant of PNPase (Fig. 1b). The levels of pnp and nlpI mRNAs in the wild type and mutant strains were quantified by qRT-PCR from cultures grown to the exponential phase of growth in Luria broth (LB). The primers used were designed to probe the pnp mRNA downstream of codon 201 and did not overlap with codon 600 of pnp. Compared to the wild-type strain, we detected enhanced expression of pnp mRNA in the pnp point mutant pnp− and no significant pnp mRNA signals in the pnp deletion mutant ∆pnp (Fig. 2a). Mitomycin C Expression of

nlpI was elevated (> 2-fold) in the pnp mutants pnp− and ∆pnp as compared to the wild-type strain (Fig. 2a). For the pnp insertion mutant pnp*, we noted no apparent alteration in either the pnp or nlpI mRNA signals (Fig. 2a).

Conversely, no alteration in pnp expression was observed when nlpI was deleted in mutant SFR319 (∆nlpI) (Fig. 2a). Combined, these observations demonstrate that the expression of nlpI is increased by mutations in pnp. However, this increase was not observed in pnp* mutant presumably because of nlpI expression being driven from the tetracycline resistance gene promoter in pnp*. This assumption would also explain detection of pnp mRNA in the pnp* mutant. To define whether the pnp–nlpI

genes are transcribed Sclareol as single click here mRNA, total bacterial RNA was first reverse-transcribed from wild-type S. Typhimurium. Standard PCR was performed using primer pairs aimed to amplify regions spanning from pnp into nlpI (Fig. 3a–c, Table S1). When combined with primers at different positions within pnp, and with a primer positioned at the 5′-end of the nlpI open reading frame (Table S1), the predicted 2.2 kb, 1 kb and 150 bp intergenic fragments were amplified from cDNA prepared from the wild-type strain MC1 (Fig. 3a–c). These observations strongly suggest that pnp and nlpI form an operon. As pnp is autoregulated by PNPase (Carzaniga et al., 2009), a pnp–nlpI operon structure would also explain the enhanced nlpI expression noted for the pnp− and ∆pnp mutants. The open reading frame for the tentative cold shock RNA helicase DeaD starts 237 bp downstream the nlpI STOP codon (McClelland et al., 2001). RT-PCR, using mRNA from wild-type S. Typhimurium as template and primers positioned within the deaD coding region, clearly detected deaD transcripts. However, using the same template, we failed to amplify any cDNA with primers positioned between the nlpI reading frame and deaD (Fig. 3d). Furthermore, as compared to the wild type, the levels of deaD mRNA remained fairly unaltered in the pnp mutant ∆pnp and ∆nlpI mutant (Fig. 2a). This suggests that deaD is transcribed independently from pnp and nlpI.

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