Abstract
Psychrophilic micro-organisms are the most dominant flora in cold habitats. Their unique ability to survive and multiply at low temperatures (<5 °C) is based on their ability to modulate the rigidity of the membrane, to transcribe, to translate and to catalyse biochemical reactions at low temperature. A number of genes are known to be upregulated during growth at low temperature and cold-inducible promoters are known to regulate the expression of genes at low temperature. In this review, we attempted to compile promoter sequences of genes that are cold-inducible so as to identify similarities and to compare the distinct features of each type of promoter when microbes are grown in the cold.
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Edited by: S. Spiro
Introduction
A large proportion of the Earth’s biosphere (>75 %) is either transiently or permanently cold, as in the polar ice caps, glaciers and deep sea where the temperature is usually low (<5 °C) (Cavicchioli & Torsten, 2000; Russell, 1998). In these cold habitats, psychrophilic micro-organisms that generally grow at an optimum temperature of 20–30 °C and also have the ability to divide at low temperatures (<5 °C) are among the most dominant flora (Baross & Morita, 1978). Studies have established that psychrophilic micro-organisms adapt to low temperature due to their ability to modulate the fluidity of the membranes, to transcribe, to translate and to catalyse biochemical reactions at low temperature (Barria et al., 2013; Singh & Shivaji, 2010; Sundareswaran et al., 2010; Uma et al., 1999). It was also demonstrated that a RNA polymerase isolated from Pseudomonas syringae (Lz4W) (Shivaji et al., 1989) had the ability to transcribe at low temperature (Uma et al., 1999) unlike the RNA polymerase of mesophilic Escherichia coli. A number of genes, including AAT in Pseudomonas syringae (Lz4W) (Sundareswaran et al., 2010), trmE in Pseudomonas syringae (Lz4W) (Singh et al., 2009), hutU in Pseudomonas syringae (Lz4W) (Janiyani & Ray, 2002), rpoS in Pseudomonas putida (Jovcic et al., 2008), deaD in Methanococcoides burtonii (Lim et al., 2000), crhC in Anabaena sp. (Chamot et al., 1999), cspI in E. coli (Wang et al., 1999), cspG in E. coli (Nakashima et al., 1996), cspB in E. coli (Lee et al., 1994), csdA in E. coli (Toone et al., 1991), cspA and cspB in Caulobacter crescentus (Mazzon et al., 2012), cspA in E. coli, and RecBCD (Goldstein et al., 1990; Pavankumar et al., 2010; Sinha et al., 2013), are known to be upregulated during cold shock by a cold-inducible promoter. In psychrophilic Pseudomonas syringae (Lz4W) it was observed that hutU is upregulated upon cold shock by a cold-inducible promoter that regulates the hutU operon. Two promoters have been recognized for hutU, with one of the promoters active at normal temperature as well as at low temperature (22 and 4 °C), whereas the another is active only at low temperature (4 °C) (Janiyani & Ray, 2002). Recently, two other cold-inducible promoters that regulate the expression of tRNA modification have been identified: GTPase (trmE) and the aspartate aminotransferase gene (AAT) in psychrophilic Pseudomonas syringae (Lz4W) (Singh et al., 2009; Sundareswaran et al., 2010). In the cold-adapted Gram-negative bacterium Pseudoalteromonas haloplanktis (TAC125), several cold-inducible promoters were isolated by cloning the TAC125 genomic DNA fragments in a shuttle vector and the promoters containing recombinant clones were selected based on their ability to express a promoterless lacZ gene (Duilio et al., 2004). Using this approach, the nucleotide sequences of several selected inserts and the transcription start sites of the transcribed mRNA were determined. A promoter consensus sequence for Pseudoalteromonas haloplanktis (TAC125) was proposed on the basis of a sequence comparison between the various active promoters (Duilio et al., 2004).
The nature of the promoter, its regulatory elements and the mechanisms of transcription at low temperature are poorly understood. Therefore, in this review, we have attempted to compile promoter sequences of genes that are cold-inducible so as to identify similarities and to compare the distinct features of each type of promoter when microbes are grown in the cold (Lisser & Margalit, 1993; Duilio et al., 2004; Singh et al., 2009).
Promoter characteristics of psychrophilic bacteria
5′ Untranslated region (UTR)
The 5′ UTR refers to the nucleotide sequence present between the transcription and translation start sites in the mRNA. Previous studies indicated that in E. coli, Anabaena sp. and Methanococcoides burtonii, the DEAD-box RNA helicase gene (deaD) and the four csp genes, which are cold-regulated, contain a 5′ UTR >100 bp in length (Fang et al., 1998; Lim et al., 2000; Janiyani & Ray, 2002; Singh et al., 2009) (Table 1). Further, in E. coli, overexpression of the 5′ UTR of cspA, cspB and csdA mRNA following cold shock induced prolonged synthesis of CspA, CspB and CsdA or simultaneous derepression of cspA, cspB and csdA (Fang et al., 1998; Jiang et al., 1996). Overexpression of the E. coli cspI 5′ UTR also caused derepression of the cold-shock response of cspI, but the derepression was weaker compared with the cspA 5′ UTR (Wang et al., 1999). The authors concluded that the optimal temperature ranges for induction of the four E. coli cold-shock-induced csp genes (cspA, B, G and I) were not the same, thereby indicating that whilst the 5′ UTR and cold-box elements may be involved in regulation of gene expression, specific sequence differences in the 5′ UTR and cold-box elements may play important roles in regulation. Thus, it is apparent that the 5′ UTR plays an important role with respect to regulation of cold-inducible genes, but studies using cspA : : lacZ fusions, which contained a variety of deletions of the 5′ UTR (Mitta et al., 1997; Yamanaka, 1999), indicated that deleting the cold-box had little effect on cold-shock induction of β-galactosidase activity and, instead, a region 11 bp upstream of the ribosome-binding site was important for translational efficiency of gene expression. These data indicate that the precise mechanism by which regulation occurs is unclear. These observations are not in accordance with the trmE gene of psychrophilic Pseudomonas syringae (Lz4W), which has one of the longest 5′ UTRs of 343 bp compared with the 5′ UTR in cold-inducible genes in other bacteria (Singh et al., 2009) (Table 1) and which has been demonstrated to be essential for regulation of cold-inducible trmE (Singh et al., 2009) based on promoter deletion experiments.
actA coding for ActA, the major virulence factor in Listeria monocytogenes, also has a long 5′ UTR of 150 bp which when deleted dramatically influenced actA expression levels (Wong et al., 2004), thus implying that secondary structural motifs within the actA mRNA 5′ UTR determine overall levels of actA expression. The 5′ UTR has also been reported to be important for mRNA stabilization following cold shock (Mitta et al., 1997). The conservation of the long 5′ UTR in low-temperature-regulated genes suggests strongly that their occurrence is more than a coincidence and supports their role in gene regulation. In most vertebrates, the 5′ UTRs are <100 bp, but in genes that are tightly controlled, the 5′ UTR is longer (Uhlmann-Schiffler et al., 2002), and such structures can modulate translation by the formation of secondary structures and by RNA–protein interaction (Gray & Wickens, 1998; Mazzon et al., 2012; Uhlmann-Schiffler et al., 2002).
Several conserved promoter elements, such as the −10 and −35 regions, cold-box, UP element, DEAD-box and Shine–Dalgarno sequence, have been reported in the 5′ UTR region (Singh et al., 2009) (Fig. 1).
Nucleotide sequence of the trmE promoter of Pseudomonas syringae (Lz4W) and its regulatory elements. The transcription start site +1 (A), −10 region (TGGATT), −35 region (TGAAAT), UP element (TACTTCTGGAAAGT), cold-box (TGAACAACTGC), DEAD-box (AACAGTGGTA), conserved region (CAAAAA), Shine–Dalgarno sequence (SDS; GAGG) and translation start site (ATG) are highlighted (Singh et al., 2009). The arrows indicate the direction of the primers, the transcription start site and the translation start site. The primer sequences are Ash 57, 5′-GCTGCAGCAAGCTACAAGTCGATGGCCC), and Ash 59, 5′-GGGGTACCCCACACCACCTCGGCCTTG.
Conserved regions (−10 and −35 region).
In bacterial promoters, two conserved regions at nucleotide positions −5 to −10 and −33 to −38 are referred to as −10 and −35 regions, respectively. The consensus sequences for the −10 and −35 regions for E. coli based on 300 genes were TATAAT and TTGACA, respectively (Lisser & Margalit, 1993). These −10 and −35 consensus sequences differed from the consensus sequences TGGATT and GGAAAT in the trmE promoter from different species of Pseudomonas, Brevibacterium linens, Arthrobacter sulfureus and Marinomonas primoryensis (Fig. 2). It was also observed that the consensus sequences for the −10 and −35 regions in the same organism varied with the gene, as was the case in trmE (Singh et al., 2009), hutU (Janiyani & Ray, 2002) and cti (Kiran et al., 2005) in Pseudomonas syringae (Lz4W). The consensus sequences for the −10 and −35 regions for Pseudoalteromonas haloplanktis were reported as TRGRTW and TATRAY, respectively (where R, A/G; Y, T/C; W, A/T; X, T/G; S, G/C) (Duilio et al., 2004). Thus, it would appear that these regions vary from organism to organism and may also depend on the gene analysed; it would be interesting to analyse further more promoters from psychrophilic organisms to support the existing results. Based on the site-specific deletion study for the trmE promoter of Pseudomonas syringae (Lz4W), it has been reported that both −10 and –35 regions are essential for transcription, and probably the −10 region is more important as deletion of this region resulted in almost total loss of promoter activity (Singh et al., 2009). It has been observed that in mesophilic bacteria such as E. coli, these two regions (−10 and −35) have a spacing of 16–18 nt (Lisser & Margalit, 1993). In contrast to the mesophilic bacteria, the psychrophilic bacteria Pseudomonas syringae (Lz4W) and Pseudoalteromonas haloplanktis have a spacing of 22 nt between these two regions (−10 and −35) (Duilio et al., 2004; Singh et al., 2009) (Fig. 2).
Comparison of the nucleotide sequence of the −10 and −35 regions of the trmE promoter of Pseudomonas syringae (Lz4W) (AM944531) with the −10 and −35 regions of the trmE promoter from Pseudomonas entomophila (CT573326), Pseudomonas fluorescens (CP000094), Pseudomonas mendocina (CP000680), Pseudomonas aeruginosa (CP000348), Pseudomonas putida (CP000926), Pseudomonas syringae (CP000075), Brevibacterium linens (AM944530), Arthrobacter sulfureus (AM944532) and Marinomonas primoryensis (AM944533). (GenBank accession numbers of the respective sequences are given in parentheses.) The consensus sequences of the −10 and −35 regions based on promoters of 11 genes from a cold-adapted bacterium Pseudoalteromonas haloplanktis (Duilio et al., 2004) and 300 different genes of E. coli (Lisser & Margalit, 1993) are also shown.
Cold-box.
Several cold-inducible genes in bacteria are known to contain a cold-box element within the 5′ UTR (Graumann et al., 1997; Graumann & Marahiel, 1998; Panoff et al., 1998; Phadtare et al., 1999; Singh et al., 2009; Thieringer et al., 1998; Yamanaka et al., 1998) and the cold-box sequences are conserved (Fig. 3). Furthermore, alignment of the cold-box sequences from cold-inducible genes of various bacteria such as E. coli, Anabaena sp., Methanococcoides burtonii, Bacillus subtilis, Brevibacterium linens, Arthrobacter sulfureus, Marinomonas primoryensis, Pseudomonas syringae (Lz4W) and other different species of Pseudomonas led to the identification of a consensus sequence for the cold-box as TGA(A/C)NAAC(T/A)G(C/A) (where N represents any nucleotide) (Fig. 3). 5′ UTR sequences with conserved elements (different to the cold-box sequence) have also been identified in cold-induced genes in Bacillus (Graumann et al., 1997).
Comparison of the nucleotide sequence of the cold-box of the trmE promoter of Pseudomonas syringae Lz4W (AM944531) with the cold-box of the trmE promoter from Pseudomonas syringae (CP000075), Pseudomonas entomophila (CT573326), Pseudomonas fluorescens (CP000094), Pseudomonas mendocina (CP000680), Pseudomonas aeruginosa (CP000438), Pseudomonas putida (CP000926), Brevibacterium linens (AM944530), Arthrobacter sulfureus (AM944532) and Marinomonas primoryensis (AM944533), and the cold-inducible genes of E. coli [cspA (AE000433), cspB (AE000252), csdA (M63288), cspG (AE000201) and cspI (AE000252)], Methanococcoides burtonii [deaD (AF199442)], Bacillus subtilis [cspB (X59715), cshA (NC000964) and cshB (NC000964)] and Anabaena sp. [crhC (AF040045)]. (GenBank accession numbers of the respective sequences are given in parentheses.) The consensus sequence is also shown.
The genes that contain the cold-box element within the 5′ UTR are regulated by cold shock, and appear to be controlled by a range of transcriptional and translational control mechanisms (reviewed by Graumann & Marahiel, 1998; Panoff et al., 1998; Phadtare et al., 1999; Thieringer et al., 1998; Yamanaka et al., 1998; Mitta et al., 1997). As noted above, the 5′ UTRs in most vertebrates are <100 bp, but are longer in genes that are tightly controlled (Uhlmann-Schiffler et al., 2002), and such structures can modulate translation by the formation of secondary structures and by RNA–protein interaction (Gray & Wickens, 1998; Uhlmann-Schiffler et al., 2002). In E. coli, deletion of the cold-box region abolished the derepression caused by overexpression of the 5′ UTR of cspA mRNA (Fang et al., 1998) indicating that derepression occurs at the level of transcription and it is probably brought about by the binding of a putative repressor to the cold-box of the mRNA of cold-inducible genes. Deletion of the cold-box region from cspA on the chromosome also caused derepression of cspA, confirming that the cold-box functions as a binding site for the putative repressor (Fang et al., 1998). Wang et al. (1999) indicated that the optimal temperature ranges for induction of the four E. coli cold-shock-induced csp genes (cspA, B, G and I) were different, thereby indicating that specific sequence differences in the 5′ UTR and cold-box elements may play important roles in regulation. Interestingly, it has also been suggested that CspE (a non-cold-shock-induced protein in the E. coli Csp family) binds to the cold-box of cspA and functions as a negative regulator of expression at 37 °C (Bae et al., 1997). These observations are not in accordance with the trmE promoter data where it has been observed that deletion of the cold-box inhibits expression of the gene (Singh et al., 2009). These data indicate that the precise mechanism by which the cold-box regulates gene expression is unclear.
UP element.
The UP element sequence was identified originally by the analysis of 31 promoters of E. coli in the region upstream to the −35 region. It is an AT-rich region (64–91 %) which is centred around the region −38 to −59 with a consensus sequence nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnn (Estrem et al., 1998). A similar UP element sequence (TACTTCTGGAAAGT) was identified in psychrophilic Pseudomonas syringae (Lz4W), which was 64.3 % AT-rich and centred around the region −41 to −54 in the trmE promoter. This UP element sequence of psychrophilic organisms is unique and it did not match with the consensus sequence of E. coli (Estrem et al., 1998). In fact, the analysis of trmE promoters from different bacterial species of Pseudomonas, Brevibacterium linens, Arthrobacter sulfureus and Marinomonas primoryensis identified the consensus UP element sequence as TACTNCTGGAAAGT (Fig. 4) (Singh et al., 2009). Previous studies have indicated that the UP element of the bacterial promoter stimulates transcription by interacting with the α-subunit of RNA polymerase (Mitta et al., 1997; Ross & Gourse, 2005). This may indeed be the case also in the case of psychrophilic Pseudomonas syringae (Lz4W) as site-specific deletion of the UP element resulted in a drastic loss of promoter activity (Singh et al., 2009).
Comparison of the nucleotide sequence of the UP element of the trmE promoter of Pseudomonas syringae (Lz4W) with the UP element of the trmE promoter from Pseudomonas entomophila (CT573326), Pseudomonas mendocina (CP000680), Pseudomonas aeruginosa (CP000438), Pseudomonas putida (CP000926), Pseudomonas syringae (CP000075), Brevibacterium linens (AM944530), Arthrobacter sulfureus (AM944532) and Marinomonas primoryensis (AM944533). (GenBank accession numbers of the respective sequences are given in parentheses.) The consensus sequence based on these nine trmE sequences is also shown.
DEAD-box.
The DEAD-box, a homologue of BoxA, is a conserved sequence AACAGTGGTA in the 5′ UTR at positions +208 to +217 in the deaD gene of Methanococcoides burtonii (Lim et al., 2000). The DEAD-box of psychrophilic Pseudomonas syringae has 70 % sequence similarity with the putative BoxA of Methanococcoides burtonii. In Pseudomonas entomophila, Pseudomonas fluorescens, Pseudomonas mendocina, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas syringae, Brevibacterium linens, Arthrobacter sulfureus and Marinomonas primoryensis, a consensus sequences for the DEAD-box has been defined as A(A/G)CAGTGGTA (Fig. 5). Site-specific deletion of the DEAD-box from the trmE promoter resulted in a drastic loss of promoter activity (Singh et al., 2009), confirming that the DEAD-box is essential in the regulation of the trmE promoter. Most probably, the DEAD-box is a transcriptional activator binding site that regulates the transcription activity of cold-inducible promoters at low temperature.
Comparison of the nucleotide sequence of the DEAD-box of the trmE promoter of Pseudomonas syringae Lz4W (AM944531) with the DEAD-box of the trmE promoter from Pseudomonas entomophila (CT573326), Pseudomonas fluorescens (CP000094), Pseudomonas mendocina (CP000680), Pseudomonas aeruginosa (CP000438), Pseudomonas putida (CP000926), Pseudomonas syringae (CP000075), Brevibacterium linens (AM944530), Arthrobacter sulfureus (AM944532) and Marinomonas primoryensis (AM944533). (GenBank accession numbers of the respective sequences are given in parentheses.) The consensus sequence is also shown.
Transcriptional silencer
A transcriptional silencer has also been identified in the promoter region of psychrophilic Vibrio sp. strain ABE-1, which plays an important role in the regulation of the cold-inducible isocitrate dehydrogenase (icdII) gene. The 35 bp transcriptional silencer sequence was identified as 5′-GTTATACCATACGGAGCTTAATTCTTTACGTAACA-3′ in the icdII promoter and it centred around the region from −560 to −526. The results from deletion studies of the transcriptional silencer indicated a 20-fold increase in expression level of the gene at low temperature (15 °C) but not at higher temperature (37 °C) (Sahara et al., 1999). Thus, the transcription silencer is another way to regulate the expression of some cold-inducible genes by its promoter.
Conclusions
Bacteria sense and transduce the low-temperature signal to a response regulator, which then induces the upregulation of genes. It is also known that bacteria preferentially transcribe cold-inducible genes at low temperature as a survival strategy. The cold-inducible promoter regulates the expression of such genes at low temperature. However, the nature of the cold-inducible promoter, its regulatory elements and the mechanisms of transcription at low temperature are poorly understood. This review has compiled all the advances on cold-inducible promoters and regulation of gene expression at low temperature. Future studies should be directed to identify a wide variety of cold-inducible promoters from different psychrophilic organisms, which would help in the creation of cold-inducible expression systems and provide a better understanding of the molecular mechanisms of cold adaptation.
Acknowledgements
A. K. S. would like to thank the Council of Scientific and Industrial Research, New Delhi, Government of India for a Fellowship. A. K. S. would also like to thank Dr Awadh Bihar Yadav (Centre of Biotechnology, University of Allahabad, India) for critical review of the manuscript.