The cold shock response is highly conserved amongst bacteria with Csps as well as PNPase also contributing to the cold Ku-0059436 manufacturer shock response in other species such as Yersinia and Bacillus (Palonen et al., 2010). The enteric pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is closely related to E. coli. It is a successful pathogen capable of infecting both warm-blooded and poikilothermic animals

including fish, nematodes, amoebas and plants (Lewis, 1975; Van der Walt et al., 1997; Charkowski et al., 2002; Cooley et al., 2003; Tenor et al., 2004; Doyel & Beuchat, 2007; Onyango et al., 2009). Hence, S. Typhimurium is also expected to experience wide fluctuations in environmental temperature. Both pnp and csdA (the latter also termed deaD) are closely linked on the genome of S. Typhimurium and only separated by nlpI that encodes for a membrane lipoprotein (Blattner et al., 1997; Ohara et al., 1999; McClelland et al., 2001; Parkhill et al., 2001; Nie et al., 2006). We have previously shown that pnp and nlpI have opposing effects on biofilm formation at decreased growth temperatures with PNPase and NlpI, respectively, enhancing and suppressing biofilm formation (Rouf et al., 2011). As nlpI is positioned between pnp and csdA, we have here investigated the contribution of pnp, nlpI and csdA (the latter hereafter referred to as deaD) in the cold

acclimatization response in S. Typhimurium. Our data show that pnp and nlpI constitute an operon that is transcriptionally separate from deaD and that PNPase, NlpI and DeaD individually Bafilomycin A1 in vivo contribute to the growth of S. Typhimurium at 15 °C. Our findings thereby define a new role for NlpI in bacterial cold acclimatization. Bacterial strains and plasmids are listed in Table 1. Bacteria were grown in Luria–Bertani medium (LB). Antibiotics (Sigma) Monoiodotyrosine were used where appropriate including ampicillin, 100 μg mL−1; chloramphenicol, 10 μg mL−1; kanamycin, 30 μg mL−1; and tetracycline, 10 μg mL−1. For induction of recombinant NlpI, media were supplemented with 0.1% L (+)-Arabinose (Sigma). Salmonella

enterica serovar Typhimurium SR-11 mutants (∆pnp, ∆nlpI and ∆deaD) were created by the one-step gene inactivation technique described previously (Datsenko & Wanner, 2000; Rouf et al., 2011). Mutated genes were transferred into S. Typhimurium SR-11 by phage P22 int transduction from S. Typhimurium ATCC 14028 background (Schmieger, 1972). The pnp–nlpI double mutant was constructed in succession. First, the PCR-amplified tetracycline resistance gene from pACYC184 was cloned into the KpnI site at codon 201 of pnp in vector pSU41. Then, the pnp::tet mutation was cloned into the pCVD442 suicide plasmid (Donnenberg & Kaper, 1991) and introduced into S. Typhimurium SR-11. The integrated vector was excised through sucrose selection to generate the pnp* mutant strain MC55.