Genetic analysis of the pnpdeaD genetic region reveals membrane lipoprotein NlpI as an independent participant in cold acclimatization of Salmonella enterica serovar Typhimurium


Correspondence: Mikael Rhen, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Box-280, SE-17 177 Stockholm, Sweden. Tel.: +46 8 52486252; fax: +46 8 330498; e-mail:


The cold acclimatization response in many bacterial species is a tightly regulated process, which ensures the correct folding of macromolecules. In enterobacteria, this response is in part dependent on polynucleotide phosphorylase, which is encoded by the gene pnp. Based on transcriptional analysis of the pnp locus of Salmonella enterica serovar Typhimurium, we show that pnp and the adjacent membrane lipoprotein nlpI gene form an operon with both genes contributing independently to the cold acclimatization response at 15 °C. Our findings thereby define a new role for NlpI in bacterial cold acclimatization.


Many microorganisms experience wide temperature fluctuations in the natural environment. As macromolecular folding strongly relies on temperature, it follows that any shift in temperature places a substantial demand on the cell in terms of the biochemical functionality (Hurme & Rhen, 1998; Klinkert & Narberhaus, 2009). Many bacteria have therefore evolved a conserved mechanism for cold acclimatization, which involves the induction of specific cold shock proteins that permit growth at lower temperatures (Phadtare et al., 1999).

In the enterobacterium Escherichia coli, the sudden transfer from 37 to 15 °C results in a response termed ‘cold shock’ that associates with a modulation in RNA turnover (Phadtare, 2004; Phadtare & Severinov, 2010). A hallmark of this response is the induction of cold shock proteins (Csps) (Phadtare et al., 1999). The major cold shock protein CspA acts as a RNA chaperone and contains a cold shock domain reminiscent of the S1 RNA binding motif. Expression of CspA itself is regulated post-translationally by temperature-dependent structural alterations in the mRNA encoding CspA (Giuliodori et al., 2010).

In addition to dedicated Csps, the cold acclimatization of E. coli requires components of the RNA degradosome, including the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase, pnp; Beran & Simons, 2001; Yamanaka & Inouye, 2001) and the proposed alternative cold shock RNA helicase CsdA (Prud'homme-Généreux et al., 2004; Turner et al., 2007). As the cold-restricted growth phenotype of E. coli csdA mutants can be complemented by plasmids encoding other proteins interacting with RNA (Awano et al., 2007), it implies that additional bacterial components may participate in the cold shock response.

The cold shock response is highly conserved amongst bacteria with Csps as well as PNPase also contributing to the cold 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 contribute to the growth of S. Typhimurium at 15 °C. Our findings thereby define a new role for NlpI in bacterial cold acclimatization.

Materials and methods

Bacterial strains, growth media and plasmids

Bacterial strains and plasmids are listed in Table 1. Bacteria were grown in Luria–Bertani medium (LB). Antibiotics (Sigma) 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).

Table 1. Bacterial strains and plasmids used in this study
Strain or plasmidRelevant genotype or descriptionSource or reference
  1. a

    Antibiotic resistance marker.

  2. b

    Nonsense codon at the beginning of RNA binding domain of the pnp reading frame.

S. enterica serovar Typhimurium
MC1Wild-type SR-11 strainClements et al. (2002)
MC55MC1 pnp::tetracyclinebRouf et al. (2011)
MC71MC1pnp−aClements et al. (2002)
SFR228MC1∆pnpRouf et al. (2011)
SFR319MC1∆nlpIRouf et al. (2011)
SFR456MC1∆deaDThis study
SFR394MC1pnp–nlpI (MC55 nlpI::kanamycinb)Rouf et al. (2011)
SFR396ATCC 14028 nlpI::kanamycinbRouf et al. (2011)
pET21cpET-based cloning and expression vector, Ampicillinb, Vector Control for pMC109Novagen®
pBAD33Arabinose regulated expression vector, Chloramphenicolb. Vector Control for pSFR04, under ara promoterGuzman et al. (1995)
pMC109pET21c::pnp, His tagged, Ampicillinb, under native promoterClements et al. (2002)
pSFR04pBAD33::nlpI, Chloramphenicolb.Rouf et al. (2011)
pSU41pACYC184-derived cloning vector, KanamycinbBartolome et al. (1991)
pCVD442The sacB containing suicide vector, AmpicillinbDonnenberg & Kaper (1991)

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 pnpnlpI 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. The nlpI::kan mutation was transferred into MC55 through P22 int phage transduction to generate pnp–nlpI double mutant strain SFR394. All mutants constructed were verified by qRT-PCR, and all pnp mutations were further verified by immunoblotting.

Immunoblot assay

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).

RNA extraction and reverse PCR

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.

Cold acclimatization assays

The ability to sustain sudden drop in growth temperature was assayed in two ways. First, bacteria were grown in LB to the exponential phase of growth at 37 °C. From that, 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.


Genetic characterization of the pnpnlpIdeaD region

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). A third mutant MC55 (pnp*) contained a tetracycline resistance gene (derived from cloning vector pACYC184) inserted at codon 201 of pnp and the resistance gene promoter reading from the same strand as pnp and nlpI (Fig. 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).

Figure 1.

(a) Genetic organization of the pnpnlpIdeaD region in the strains as adapted from the Salmonella enterica serovar Typhimurium LT2 genome sequence (McClelland et al., 2001). (b) Expression of PNPase as determined by immunoblotting from the wild-type strain (lane 1), pnp lacking the S1 domain (lane 2), ∆pnp lacking pnp (lane 3), ∆nlpI lacking nlpI (lane 4), the pnpnlpI double mutant (lane 5), pnp* containing pnp::tet (lane 6).

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). 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.

Figure 2.

Expression of pnp and nlpI transcripts in mutants (a) or complemented mutants (b) compared to the wild type during growth in LB media as assessed by qRT-PCR. The rfaH served as endogenous control as specified in Rouf et al. (2011).

To define whether the pnp–nlpI genes are transcribed as single 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.

Figure 3.

Co-transcriptional analysis of pnp and nlpI genes in ethidium bromide–stained 1% agarose gel revealing PCR-amplified pnpnlpI intergenic products using wild-type genomic DNA (lane 1), cDNA (lane 2), RNA template (lane 3) or ∆pnp genomic DNA as template (lane 4). The primer pairs used were RTF1/RTR1 (a), RTF2/RTR1 (b) or RTF3/RTR1 (c). (d) Co-transcriptional analysis of nlpI and deaD genes in ethidium bromide–stained 1% agarose gel revealing PCR-amplified nlpI-deaD intergenic products using wild-type genomic DNA (lane 1, 5), cDNA (lane 2, 6), RNA (lane 3, 7) or ∆deaD genomic DNA as template (lane 4, 8) with FdeaD/RdeaD (lane 1–4) or RTF4/RTR2 (lane 5–8) as primer pairs.

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.

Mutational inactivation of either pnp, nlpI or deaD results in impaired growth at 15 °C

The ability of the pnp, nlpI and deaD mutants to acclimatize and grow when shifted from 37 to 15 °C was determined in broth culture. In this assay, all the three pnp mutants revealed a comparable retardation in growth (Fig. 4a). As the expression of nlpI was not diminished in any of the three pnp mutants (Fig. 2a), the impaired cold acclimatization could not originate from nlpI, and so in subsequent experiments, we only used the ∆pnp mutant (SFR228).

Figure 4.

Cold acclimatization of pnp, nlpI and deaD mutants. (a–d) Growth in Luria broth showing the ability of the strains to resume growth after a 37–15 °C temperature shift by subculture from log phase of wild type and the mutants.

The ∆nlpI mutant also showed a reduced ability to grow at 15 °C comparable to the pnp mutants (Fig. 4a). As pnp expression was unaffected in this mutant, it infers that pnp and nlpI contribute individually to growth of S. Typhimurium at 15 °C. Further evidence to support this view was the observed slower growth for the pnp–nlpI double mutant (SFR394) (Fig. 4a).

Complementation of pnp (pMC109) or nlpI (pSFR04) in the respective single mutants almost restored normal growth at 15 °C (Fig. 4b). However, the introduction of either pMC109 or pSFR04 into the pnp–nlpI double mutant resulted in only a partial restoration of growth at 15 °C. An almost complete restoration of growth was achieved when the pnp–nlpI double mutant was complemented with both nlpI and pnp (Fig. 4c).

A second cold acclimatization assay was performed comparing growth on Luria agar plates incubated at either 15 or 37 °C. The ∆pnp, ∆nlpI and pnp–nlpI double mutants were assayed, and the results were comparable to the broth assay at 15 °C (Figs 4 and 5). The ∆pnp and ∆nlpI mutants both showed a restricted recovery when transferred to 15 °C (Fig. 5). In this assay, the growth defect of the pnp–nlpI double mutant appeared more pronounced in relation to either of the single mutants (Fig. 5). Similar to the broth assay, we also observed a restoration of growth when single mutants were complemented with the plasmids expressing the respective pnp or nlpI genes (Fig. 5). However, to restore any significant growth in the pnp–nlpI double mutant, plasmids coding for both pnp and nlpI had to be introduced (Fig. 5).

Figure 5.

Luria agar plate (after 36 h) showing the ability of wild-type Salmonella enterica serovar Typhimurium and mutants to resume growth after a 37 to 15 °C temperature shift at drop-on-lawn culture plates.

The contribution of the deaD gene to the cold acclimation response was also determined by observing the growth of mutant SFR456 (∆deaD) in both the broth and plate assays. The mutant revealed a marked growth defect at 15 °C (Figs 4d and 5), but this growth defect was, however, not complemented by either pnp or nlpI.


Altered folding as well as a controlled degradation and stabilization of ribonucleic acids constitutes important elements of bacterial adaptation to altered temperatures (Hurme & Rhen, 1998; Giuliodori et al., 2010). Such alterations also include helicases, RNA chaperons and ribonucleases (Phadtare & Severinov, 2010). Alterations in RNA folding may furthermore act as an endogenous post-transcriptional control of gene expression (Beran & Simons, 2001; Giuliodori et al., 2010).

Expression and post-transcriptional regulation of PNPase have been thoroughly detailed in E. coli and serve as a model for temperature-associated post-translational gene regulation. Transcription of E. coli pnp is initiated from two promoters. The first directs expression of the immediate upstream gene rpsO, and the second is positioned in the rpsO-pnp intergenic region (Portiers & Reginer, 1984). Irrespective of the transcriptional start site, the pnp mRNA is vulnerable to cleavage by endoribonuclease RNase III at positions within 75 nucleotides upstream the pnp ORF, which in turn initiates degradation of the pnp mRNA by PNPase itself (Portier et al., 1987). Upon a cold shock, the pnp mRNA becomes stabilized allowing enhanced expression of PNPase (Beran & Simons, 2001).

In enterobacteria, pnp is followed by nlpI (Blattner et al., 1997; McClelland et al., 2001; Nie et al., 2006). For E. coli, NlpI has been shown to be a lipoprotein (Ohara et al., 1999). We recently demonstrated that PNPase and NlpI posed opposing effect on biofilm formation in S. Typhimurium at decreased growth temperature (Rouf et al., 2011). Experiments that followed here demonstrate that mutational inactivation of pnp in S. Typhimurium results in an expected restricted growth at 15 °C. In addition, the experiments showed that pnp transcripts continued into nlpI and that nonpolar pnp mutations increased nlpI expression. Although S. Typhimurium pnp and nlpI are separated by 109 base pairs, the promoter prediction software bprom ( failed to define any tentative nlpI promoter within this intergenic region (data not shown). Combined with the gene expression analysis, this strongly suggests that pnp and nlpI form an operon and implies that nlpI is subject to the same post-translational regulation of pnp. However, we cannot formally exclude potential nlpI promoters within pnp.

The co-transcription of pnp and nlpI led us to detail whether, and to what extent, NlpI contributed to cold acclimatization. The data presented in this study demonstrate that nlpI does indeed functionally act as a cold shock gene in concert with, but independently of, pnp. Evidence to support includes the observation that two of the three pnp mutants applied in this study had enhanced expression of nlpI, whilst the third had unaffected nlpI mRNA levels compared to the wild type, yet all three mutants showed a very similar defect for growth at 15 °C. In addition, a pnp–nlpI double mutant had more restricted growth at 15 °C compared to either single mutant, whilst cloned pnp and nlpI enhanced the replication of all the respective mutants at 15 °C (Figs 4b and 5).

The nlpI gene is adjacent to csdA/deaD in the genomes of enterobacteria (Blattner et al., 1997; McClelland et al., 2001; Nie et al., 2006). The csdA gene encodes for an alternative RNA helicase that in E. coli also contributes to cold acclimatization (Turner et al., 2007). In S. Typhimurium, the homologue for csdA is defined as deaD. Deleting deaD in S. Typhimurium resulted in a cold-sensitive growth phenotype. However, we could not trans-complement the cold-restricted growth of the deaD mutant phenotype with either pnp or nlpI. This demonstrates specificity of the complementation by either pnp or nlpI. We also could not detect transcripts spanning the nlpI and deaD reading frames, whilst the promoter prediction software bprom was able to identify a promoter region in the region separating nlpI from deaD with a high predicted probability (data not shown), suggesting that they are transcribed separately.

To summarize, our observations imply that pnp and nlpI form a transcriptionally linked region, followed by deaD, and that all three genes individually contribute to cold acclimatization in S. Typhimurium. Furthermore, our results showed that apart from dedicated gene regulatory circuits and chaperones, cold acclimatization in S. Typhimurium also significantly relies on an outer membrane protein NlpI.


This study was supported by the Swedish Medical Research Council. S.F.R is a PhD fellow from IRTG 1273 funded by the German Research Foundation, and N.A. is a PhD fellow of HEC, Pakistan.