Absolute requirement for polyamines for growth of Escherichia coli mutants (mnmE/G) defective in modification of the wobble anticodon of transfer-RNA

Abstract

The genes mnmE and mnmG are responsible for the modification of uridine 34, ‘the wobble position’ of many aminoacyl-tRNAs. Deletion of these genes affects the strength of the codon-anticodon interactions of the aminoacyl-tRNAs with the mRNAs and the ribosomes. However, deletion of these genes does not usually have a significant effect on the growth rate of the standard Escherichia coli strains. In contrast, we have found that if the host E. coli strain is deficient in the synthesis of polyamines, deletion of the mnmE or mnmG gene results in complete inhibition of growth unless the medium contains polyamines. The finding of an absolute requirement for polyamines in our current work will be significant in studies on polyamine function, in studies on the function of the mnmE/G genes, and in studies on the role of aminoacyl-tRNAs in protein biosynthesis.

Escherichia coli mutants (mnmE/G) defective in transfer-RNA modification need polyamines for growth.

INTRODUCTION

Polyamines (such as putrescine, spermidine or spermine) are highly abundant in essentially all organisms ranging from bacteria to humans and have been implicated in many biological processes, including nucleic acid and protein synthesis, cell growth, binding to membrane phospholipids, N-methyl-D-aspartate receptors and protection against oxygen toxicity. (Reviewed by Tabor and Tabor 1984; Cohen 1998; Igarashi and Kashiwagi 2006; Pegg and Casero 2011; Michael 2016; and Pegg 2016).

The work in this laboratory has largely been concerned with the functions of polyamines in Escherichia coli. For these studies, we had inactivated the genes involved in the biosynthesis of polyamines, and found that these strains, which lack detectable polyamines, grow indefinitely in purified medium, albeit at ca 50%–60% of the growth rate of strains that are not polyamine-deficient (Chattopadhyay, Tabor and Tabor 2009). However, these polyamine mutants are sensitive to abiotic stresses including oxidative stress (Minton, Tabor and Tabor 1990; Chattopadhyay, Tabor and Tabor 2003) and acid stress (Jung and Kim 2003; Chattopadhyay and Tabor 2013; Chattopadhyay et al.2015).

There is a large body of literature, ever since the initial work of Elseviers’ group (Elseviers, Petrullo and Gallagher 1984) on modification of aminoacyl-transfer RNAs (aminoacyl-tRNA) by mnmE and mnmG¹ encoded proteins. These genes are responsible for the modification of uridine 34, ‘the wobble position’ of many aminoacyl-tRNAs (Fig. 1). Deletion of these genes affects the strength of the codon-anticodon interactions of the aminoacyl-tRNAs with mRNA and with the ribosomes (Armengod et al.2012). Inactivation of the mnmE/G pathway has been described as producing a pleiotropic phenotype in bacteria as well as mitochondrial dysfunction in human cell lines (Armengod et al.2012 and Armengod et al.2014).

Figure 1.

mnmE: mnmG mediated tRNA modification (First reported by Armengod et al, 2012).

Our earlier studies on the role of polyamines in increasing translation of an amber codon in vivo (Tabor and Tabor 1982) and on the effect of polyamines on ribosomal frameshifting in yeast (Balasundaram et al.1994), along with our more recent work on the acid resistance of E. coli, led us to the Foster group's (Gong, Ma and Foster 2004) discovery that the acid resistance of E. coli requires an intact mnmE (trmE) gene. With numerous studies showing interactions of polyamines and tRNA (reviewed by Lightfoot and Hall 2014), it seemed reasonable that the mnmE and mnmG genes would be of interest in our current work on polyamines and the acid response system. In the current paper we show that in strains containing deletion of either the mnmE or mnmG gene, the growth of the subsequent E. coli cells is completely dependent upon the presence of polyamines.

MATERIALS AND METHODS

Strain constructions

All strains were constructed on the parent of Keio collection (BW25113; CGSC 7636). The strains used for the P1 transduction were obtained from the Keio collection from the Yale Univ. E. coli Genetics Center. The deleted genes in this collection contain a kanamycin insert, which was used for the selection process. For use in subsequent transduction experiments, the kanamycin insert was excised from the transduced strain by the FLP recombinase, as described by (Baba et al.2006) and by (Datsenko and Wanner 2000). P1 transductions were carried out essentially as described by (Miller 1992).

The E. coli strains and their source are listed in Table 1. HT779 is the parent strain and has no deletions in the genes involved the polyamine biosynthesis pathway or in the mnmE or mnmG genes. HT873 is a derivative of HT779 that we constructed by sequential deletion of the genes involved in the polyamine biosynthetic pathway (Chattopadhyay et al.2015). The resultant strain contains no polyamines but can grow indefinitely in polyamine-free medium, albeit at 50%–60% of the growth rate of HT779 (Fig. 2A).

Table 1.

All the strains used for this study were obtained from the Yale E. coli Genetic Stock Center (Keio collection (Baba et al.2006)).

Strain	Relevant genotype
HT779	speA + speC + speD + ldcC + speF + adiA + cadA + mnmE + mnmG+	Parent of Keio collection (BW25113; CGSC 7636)
HT949	speA + speC + speD + ldcC + speF + adiA + cadA + ΔmnmE mnmG+	ΔmnmE From Keio collection JW3084–1; CGSC 10 693
HT950	speA + speC + speD + ldcC + speF + adiA + cadA + mnmE + ΔmnmG	ΔmnmG From Keio collection JW3719–1; CGSC 11 677
HT873	ΔspeA ΔspeC ΔspeD ΔldcC ΔspeF ΔadiA ΔcadA mnmE + mnmG+	Seven sequential gene deletions of polyamine biosynthetic pathway in HT779 strain. Constructed by P1 transduction from deletion strains in Keio collection.
HT880	ΔspeA ΔspeC ΔspeD ΔldcC ΔspeF ΔadiA ΔcadA ΔmnmE	ΔmnmE from Keio collection transduced into HT873
HT884	ΔspeA ΔspeC ΔspeD ΔldcC ΔspeF ΔadiA ΔcadA ΔmnmG	ΔmnmG from Keio collection transduced into HT873

Figure 2.

Absolute inhibition of growth if the polyamine-deficient mutant also contains a deletion in the t-RNA modification pathway. (A) A strain with a deleted polyamine biosynthetic pathway (HT873) grew well, albeit at a slightly slower rate than the parent strain (HT779) (B) Deletion of the mnmE gene (HT880) or (C) Deletion of the mnmG gene (HT884) resulted in complete halt of growth of the E. coli cells in the absence of amines.

Culture conditions and aerobic growth

Cultures were grown in polyamine-free Vogel-Bonner minimal medium (VBC) containing 0.4% glucose at 37°C with shaking (Vogel and Bonner 1956). Colonies (usually two) from LB plates were suspended in 45 ml VBC/glucose medium and incubated with occasional dilutions in the same minimal medium for about 24–36 hours at 37°C to deplete the polyamines present in the original colonies. The cultures were then diluted in VBC/glucose to an optical density at 600 nm of ca. 0.05 and incubated at 37°C, with additional dilutions so that the overnight optical densities would still be in the readable range (OD < 1). The optical density data in the graphs have been corrected for these dilutions.

RESULTS

The polyamine-deficient strain (HT873) grows indefinitely in the polyamine-deficient VBC medium albeit at a slightly lower growth rate 50%–60% (Fig. 2A). However, if the polyamine-deficient strain also contains a deletion of either mnmE (HT880) or mnmG (HT884) growth is completely inhibited (Fig. 2B and C). After much longer incubation in the polyamine deficient medium (for several days), growth was noted, consistent with the development of bypass mutations (data not shown).

Supplementation with putrescine or spermidine restored the growth of both HT880 (Fig. 3A and B) and HT884 (Fig. 3C and D). Either 10^–4 M putrescine or 10^–4 M spermidine restored optimum growth, after a brief lag period. Similar results were obtained via the addition of the diamine cadaverine (data not shown). The importance of polyamines was further confirmed by a growth experiment (Fig. 3E) showing that deletion of either mnmE or mnmG does not have a significant effect on the growth rate of the strain (HT779) that has an intact polyamine biosynthetic pathway.

Figure 3.

Added polyamines or intact polyamine biosynthesis pathway can overcome the growth inhibition caused by deletion of the mnmE gene (HT880- A, B) or the mnmG gene (HT884- C, D) Polyamines were added to the inhibited cultures and the optical densities at 600 nm (corrected for dilutions) were measured with time. (E)- Deletion of the mnmE gene or of the mnmG gene does not inhibit the growth rate of a strain (HT779) with an intact polyamine biosynthetic pathway.

DISCUSSION

We postulate that polyamines enhance productive anticodon-codon pairing when it is compromised by the absence of wobble position modification generated by MnmE or MnmG, and thus directly and indirectly affect the structure of aminoacyl-tRNA and various components of the ribosome-protein synthesizing complex. This speculation is consistent with the many studies on the interaction of polyamines and tRNA (reviewed by Lightfoot and Hall 2014) and with two extensive studies by Farabaugh and his group showing the importance of codon-anticodon strength on the translational accuracy (Manickam et al.2014; Manickam et al.2016) and of (Hetricket et al.2010) showing that polyamines accelerate codon recognition by tRNAs on the ribosome. These observations including our current in vivo studies are supported by evidences from other laboratories, where they have found that polyamines bind and modulate the structure of tRNAs in vitro (Frydman, de los Santos and Frydman 1990). Hori and Oshima's group (Terui et al.2005; Hori et al.2018) have also reported that different branch chain polyamines are necessary for the survival of thermophilic bacteria, particularly if they are missing genes for tRNA modification pathway. As pointed out in the review of (Lightfoot and Hall 2014), the effects of polyamines are distinct from the effects of divalent cations, such as Mg²⁺. It has also been reported that polyamine binds to 11 distinct sites of tRNA in vitro and Mg²⁺ can replace polyamines for some of the sites, but not all (Quigley, Teeter and Rich 1978; Frydman, de los Santos and Frydman 1990; Lightfoot and Hall 2014). Thus, the role of polyamines in binding and stabilizing is mostly independent of its polycationic nature. This is also consistent with our finding that the polyamine deficient mnmE/G mutants are unable to grow in purified medium, even though the medium contains MgSO₄ (810.0 µM). Our studies did not show an effect on growth of the cells upon addition of higher concentrations of MgSO₄ (data not shown). Although, our hypothesis that polyamines help in stabilizing codon-anticodon interactions in the mnmE/G mutants is not supported by any mechanistic studies, this fundamental study is supported by other in vitro and in vivo observations. Our experiments, also do not exclude a more indirect effect of a polyamine deficiency on the mnmE/G modification system.

From these results, it seems that polyamines are not only required for survival of polyamine deficient E. coli during various abiotic stresses as mentioned in the introduction but are also required for in vivo maintenance of proper codon-anticodon interactions if the mnmE/G system is absent. Thus, the finding of an absolute requirement for polyamines in our current work is significant both in contributing to our understanding of polyamine function but also in demonstrating that the role of polyamines has to be considered in studies on the physiologic role of mnmE and mnmG genes.

The strain used in this paper is unique and particularly useful in that it shows an absolute requirement for polyamines in a strain with both well-defined mutations in the genes responsible for the biosynthesis of polyamines and in the mnmE/G genes that are responsible for the modification of aminoacyl-tRNAs.

Notes

From the Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland

FUNDING

This research was supported by the Intramural Research Program of the National Institutes of Health (National Institute of Diabetes, Digestive and Kidney Diseases).

Conflict of interest . None declared.