All mutants constructed were verified by qRT-PCR, and all pnp mutations were further verified by immunoblotting. Immunoblotting of SDS–PAGE gels was performed as outlined in QIAexpress® Detection Assay Handbook (Qiagen) using polyvinylidene difluoride membranes (HybondTM P; Amersham). A polyclonal rabbit anti-PNPase serum, generated by immunizing rabbits with purified PNPase (Clements et al., 2002) under
the ethical permit Dnr 79/2001, was used as primary antibody (1 : 500), whilst horseradish peroxidase–conjugated goat anti-rabbit-IgG (1 : 5000, Pierce) was applied as secondary antibody. Blots were analysed using SuperSignal detection kit (Pierce) and ChemiDoc XRS system (Bio-Rad). Smad pathway For extraction of RNA, bacteria were grown in LB to the exponential phase of growth. Total bacterial RNA was isolated using the TRI Reagent (Sigma). To determine transcripts spanning the pnp–nlpI and nlpI–deaD genes, RNA samples were first reverse-transcribed using the M-MLV kit (Invitrogen).
Standard PCR was then carried out on the cDNA products using different sets of primers (Supporting Information, Table S1). Total genomic DNA was isolated using the Wizard Genomic DNA Purification kit (Promega) and used as a positive template control. DNA fragments were analysed on ethidium bromide–stained agarose gels. RNA quantification was carried out by quantitative reverse PCR using the SYBR Green JumpStart kit (Sigma) and the ABI Prism 7000 sequence detection system with primers detailed in Table S1. The ability to sustain sudden drop in growth temperature was assayed in two ways. First, bacteria were grown in LB to GSK458 research buy the exponential phase of growth at 37 °C. From that, Rucaparib molecular weight 100 μL of the culture was adjusted to an OD600 nm of 0.5, which then was inoculated into 50 mL of precooled LB on a shaker at 15 °C. Optical densities of the cultures were then followed at regular time intervals. In the second assay, bacteria were grown
over night in LB at 37 °C and diluted in phosphate-buffered saline to an OD600 nm of 0.5. Subsequent 1 : 10 serial dilutions were then inoculated in 10 μL drops on two separate LB agar plates. One was incubated at 37 °C and the other at 15 °C. In S. Typhimurium, pnp and nlpI are linked and read from the same DNA strand (McClelland et al., 2001). The pnp STOP codon and the nlpI START codons are separated by 109 nucleotides (McClelland et al., 2001; Fig. 1a). Because of the close proximity of pnp to nlpI, we examined to what extent mutations in pnp would affect expression of nlpI. We compared the pnp and nlpI mRNA levels in the wild-type S. Typhimurium SR-11 (MC1) and three pnp mutants. These mutants were MC71 (pnp−) expressing a truncated PNPase because of a point mutation replacing codon 600 with stop codon TAA and mutant SFR228 (∆pnp) having the entire pnp open reading frame deleted (Fig. 1a) (Clements et al., 2002; Rouf et al., 2011).