The i6A37 tRNA modification is essential for proper decoding of UUX-Leucine codons during rpoS and iraP translation.

The translation of rpoS(σ(S)), the general stress/stationary phase sigma factor, is tightly regulated at the post-transcriptional level by several factors via mechanisms that are not clearly defined. One of these factors is MiaA, the enzyme necessary for the first step in theN(6)-isopentyl-2-thiomethyl adenosinemethyl adenosine 37 (ms(2)i(6)A37) tRNA modification. We tested the hypothesis that an elevated UUX-Leucine/total leucine codon ratio can be used to identify transcripts whose translation would be sensitive to loss of the MiaA-dependent modification. We identified iraPas another candidate MiaA-sensitive gene, based on the UUX-Leucine/total leucine codon ratio. AniraP-lacZ fusion was significantly decreased in the abse nce of MiaA, consistent with our predictive model. To determine the role of MiaA in UUX-Leucine decoding in rpoS and iraP, we measured β-galactosidase-specific activity of miaA(-)rpo Sandira P translational fusions upon overexpression of leucine tRNAs. We observed suppression of the MiaA effect on rpoS, and notira P, via overexpression of tRNA(LeuX)but not tRNA(LeuZ) We also tested the hypothesis that the MiaA requirement for rpoS and iraP translation is due to decoding of UUX-Leucine codons within the rpoS and iraP transcripts, respectively. We observed a partial suppression of the MiaA requirement for rpoS and iraP translational fusions containing one or both UUX-Leucine codons removed. Taken together, this suggests that MiaA is necessary for rpoS and iraP translation through proper decoding of UUX-Leucine codons and that rpoS and iraP mRNAs are both modification tunable transcripts (MoTTs) via the presence of the modification.

INTRODUCTION

Transfer RNA (tRNA) modifications play a critical role in the promotion of translation fidelity (Urbonavicius et al. 2001). The absence of tRNA modifications is known to increase the frequency of translational frameshifting (Björk et al. 1999; Urbonavicius et al. 2001, 2003). There are a host of tRNA modifications that are most crucial in translational frameshift suppression and the majority of them reside within the anticodon stem–loop (ASL), flanking the anticodon at positions 34 and 37, including 5-methyluridine (m5U34), 5-methylcytidine (m5C34), and N6-isopentenyladenosine (i6A37) (Endres et al. 2015). Certain tRNA modifications act in a regulatory manner on physiological circuits within the cell, such as DNA damage and oxidative stress in eukaryotic cells (Begley et al. 2007; Chan et al. 2010, 2012; Patil et al. 2012; Lamichhane et al. 2013a). There is limited information on the regulatory role of tRNA modifications in bacterial cells. We previously identified an important role for the Escherichia coli ms2i6A37 synthesis enzyme, MiaA, in the translation of rpoS mRNA (Thompson and Gottesman 2014).

RpoS (or σS) is the stationary phase/general stress response alternative sigma factor necessary for the adaptation of bacterial cells to the stationary phase environment (Hengge-Aronis 1993, 1996). RpoS contributes to stationary phase homeostasis by initiating the transcription of a large subset of genes that respond to limiting nutrients and increased exposure to reactive oxygen intermediates (Hengge-Aronis 1993; Tanaka et al. 1993). Since RpoS levels are important for the stationary phase stress response, RpoS levels are tightly regulated at the transcriptional level and at post-transcriptional levels (Lange et al. 1995; Brown and Elliot 1996; Hirsch and Elliott 2002). RpoS translation is stimulated by the three Hfq-dependent small regulatory RNAs: DsrA, RprA, and ArcZ in response to different environmental signals (Sledjeski et al. 1996; Majdalani et al. 1998, 2002; Mandin and Gottesman 2010). RpoS is also regulated at the level of protein stability by the ATP-dependent protease ClpXP, with the assistance of RssB, which serves as an adaptor protein (Gottesman 1996; Zhou and Gottesman 1998). There are three anti-adaptor proteins, IraP, IraD, and IraM, which all prevent RssB interaction with RpoS under different conditions and increase RpoS stability as a consequence (Bougdour et al. 2006, 2008).

In addition to stimulation of translational initiation by several small regulatory RNAs and regulation of RpoS stability, translation of the rpoS open reading frame (ORF) is also regulated by cis and trans acting elements. For example, SsrA is necessary for rpoS translation through a mechanism that is not completely understood (Ranquet and Gottesman 2007). The presence of rare codons within the rpoS ORF also positively contributes to mRNA stability by decreasing degradation by RNaseE (Kolmsee and Hengge 2011). Additionally, AceE, a subunit of pyruvate dehydrogenase, also influences rpoS ORF translation via an undefined mechanism (Battesti et al. 2015). Finally, the MiaA tRNA modification enzyme is required for efficient translation of the rpoS ORF translation (Thompson and Gottesman 2014).

MiaA is required for the first step in the formation of ms2i6A37 modification on tRNAs that read UUX codons (Bartz et al. 1970). Specifically, MiaA catalyzes the addition of the isopentyl group (i6) to A37 of these tRNAs (Bartz et al. 1970). MiaB then catalyzes the addition of a methylthio group (ms2) to complete the ms2i6A37 tRNA modification (Vold et al. 1979; Esberg et al. 1999). While MiaB is necessary for completing the ms2i6A37 tRNA modification, our initial analysis of the effect of tRNA modifications on rpoS suggested that MiaB was not necessary for full rpoS expression (Thompson and Gottesman 2014). Therefore, this work is focused on characterizing the MiaA effect on rpoS. MiaA is necessary for expression of genes involved in the biosynthesis of amino acids in Escherichia coli and Salmonella enterica subspecies enterica servar Typhimurium, including tryptophan and phenylalanine in E. coli as well as leucine in S. typhimurium (Gowrishankar and Pittard 1982; Blum 1988). The MiaA requirement for expression of the tryptophan and phenylalanine operons is due to its role in modulating transcriptional attenuation (Landick et al. 1990; Pages and Buckingham 1990). We hypothesized that leucine codon usage may play a role in the requirement for MiaA in the efficient translation of rpoS, due to use of UUX-Leucine rather than CXX-Leucine codons within the rpoS ORF (Thompson and Gottesman 2014).

Here, we test this hypothesis, both extending our previous tests with rpoS to another UUX-rich gene, iraP. We demonstrate the ability to identify putative ms2i6A37 modification tunable transcripts (MoTTs), via MiaA sensitivity, for a given gene based on UUX-Leucine codon usage for that gene (Endres et al. 2015). We hypothesize that genes for ORFs with high UUX Leucine codon usage, or HULC proteins, defined as having a UUX-Leucine codon usage ratio >0.22, likely have i6A37 MoTTs. We show that iraP has a similar enrichment ratio for UUX-Leucine codons as rpoS and that its translation requires the i6A37 tRNA modification catalyzed by MiaA. Furthermore, we demonstrate that UUX-leu to CUX-leu codon substitutions within rpoS and iraP can suppress the effects of ΔmiaA mutants on the translation of these ORFs, suggesting that MiaA is required for rpoS and iraP translation, at least in part, for efficient decoding of UUX-Leucine codons in these two genes.

RESULTS

Replacing MiaA-sensitive leucine codons (UUGs and UUAs) with MiaA-insensitive leucine codons (CUUs and CUCs) partially suppresses the MiaA requirement for rpoS synthesis

We previously used an arabinose-inducible rpoS990-lacZ translational fusion to demonstrate the role of the MiaA-catalyzed i6A modification on rpoS synthesis (Thompson and Gottesman 2014). The P-rpoS990-lacZ translation fusion contains the entire rpoS ORF, except for the termination codon; the small-RNA responsive 5′ UTR of rpoS and the native promoter are replaced by the araBAD promoter (P) (Fig. 1B; Supplemental Table S1; Thompson and Gottesman 2014). All P-rpoS990-lacZ translation fusion experiments were executed in an rssB− background to rule out effects on rpoS degradation.

FIGURE 1.

Translation of leu*3 alleles of rpoS and iraP in the absence of ms2i6A37 tRNA modification. (A) The rssB− miaA+ (KMT33001) and rssB− miaA− (KMT33002) P-rpoS990(leu*3)-lacZ translational fusion strains, as well as rssB− miaA+ (KMT30003) and rssB− miaA− (KMT30011A) P-rpoS990-lacZ translational fusion strains, were assayed for β-galactosidase-specific activity following arabinose induction. β-galactosidase-specific activity is defined as the slope of OD420 of the collected sample cell lysate divided by the OD600 of the collected sample of the culture, as described in Materials and Methods. Each value represents the mean of at least three replicate experiments; the error bars represent the standard error of the mean (SEM). (B) Schematic comparison of the P-rpoS990-lacZ and P-rpoS990(leu*3)-lacZ translational fusions used in this experiment. The stars within the P-rpoS990(leu*3)-lacZ translational fusion represent silent leucine mutations where rare leucine codons (UUA and UUG) were changed to abundant leucine codons (CUU or CUC). (C) The miaA+ (KMT45000) and miaA− (KMT45002) P-iraP258(leu*3)-lacZ translational fusion strains, as well as miaA+ (KMT42000) and miaA− (KMT42002) P-iraP258-lacZ translational fusion strains were assayed for β-galactosidase-specific activity following arabinose induction. Each value represents the mean of at least three replicate experiments; the error bars represent the standard error of the mean (SEM). (D) Schematic comparison of the P-iraP258-lacZ and P-iraP258(leu*3)-lacZ translational fusions used in this experiment. The green stars within the P-iraP258(leu*3)-lacZ translational fusion represent silent leucine mutations where rare leucine codons (UUA and UUG) were changed to abundant leucine codons (CUU or CUC). Each value represents the mean of at least three replicate experiments; the error bars represent the standard error of the mean (SEM).

We previously hypothesized that the greater than expected ratio of MiaA-sensitive leucine codon to total leucine codon usage within the rpoS ORF suggested that MiaA may function to ensure proper decoding of these UUX-Leucine codons within rpoS (Thompson and Gottesman 2014). To test this hypothesis, we constructed a P-rpoS990-lacZ translational fusion in which the first and wobble position of all MiaA-sensitive leucine codons (UUAs and UUGs) in rpoS were changed to create MiaA-insensitive leucine codons (CUUs and CUCs), creating a series of silent leucine mutations throughout the rpoS990 region of the translational fusion (Table 1, leu*3; Fig. 1B). The rationale behind the selection of CUU and CUC codons is that they are present at frequencies similar to UUA and UUG codons (http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/in-vitro-genetics/codon-usage.html). There are six codons that code for leucine, UUR, and CUX. The average frequency of decoding for leucine by CUU and CUC codons are 0.10, which is similar to the 0.11 frequency seen with UUA and UUG codons. In contrast, CUA and CUG Leu codons have average frequencies of 0.03 and 0.55, respectively.

TABLE 1.

Silent leucine mutations in the rpoS leu* alleles

We then measured the β-galactosidase-specific activity (as defined in Materials and Methods) as the slope of OD420/OD600 of the wild-type and leu*3 fusions in rssB− miaA+ and rssB− miaA− genetic backgrounds (Fig. 1A). There was an exponential increase in the β-galactosidase-specific activity of both the wild-type P-rpoS990-lacZ and P-rpoS990(leu*3)-lacZ translational fusions in the presence of MiaA (Fig. 1A). As previously reported, the absence of MiaA resulted in severely defective induction of the P-rpoS990-lacZ in comparison to the miaA+ strain (Fig. 1A). Specifically, 25 min after arabinose induction (t25), there was a fivefold decrease in P-rpoS990-lacZ expression in the absence of MiaA (Fig. 1A). The induction of the P-rpoS990(leu*3)-lacZ translational fusion was not as defective in the absence of MiaA in comparison to the wild type (1.5-fold decrease in P-rpoS990[leu*3]-lacZ) expression in the absence of MiaA at t25 (compared to fivefold in the wild-type fusion). This suggests that the presence of UUX-Leucine codons, which are sensitive to MiaA-modified leucine tRNAs, contribute to the requirement of MiaA for full rpoS synthesis. However, the fact that the removal of the UUX-Leucine codons did not result in full suppression of the MiaA requirement for rpoS synthesis suggests that MiaA does more for rpoS translation than just ensuring that UUX-Leucine tRNAs properly recognize their cognate codons. MiaA-sensitive codons also consist of UUX phenylalanine, UCX serine, UAX tyrosine, UGX cysteine, and UGG tryptophan. It is possible that these codons may not be sufficiently decoded in the absence of MiaA as well. Also, the expression from the miaA+ P-rpoS990(leu*3)-lacZ translational fusion was slightly decreased in comparison to the miaA+ P-rpoS990-lacZ, suggesting that the UUX-Leucine codons are required for optimal rpoS translation in the presence of MiaA.

The translation of iraP requires the ms2i6A37 tRNA modification for decoding of MiaA-sensitive leucine codons

To determine if the ms2i6A37 modification was required for optimal translation of other HULC proteins, we examined the leucine codon usage of several arbitrarily selected genes related to rpoS regulation. In addition, we acquired a table documenting the leucine codon usage of every ORF in the E. coli genome (Supplemental Table S7). We decided to test our hypothesis on iraP, which encodes an anti-adaptor that plays a role in rpoS stability (Bougdour et al. 2006). iraP is a HULC protein, with a UUX-Leucine codon usage ratio of 0.46, which is twofold greater than the expected UUX-Leucine codon ratio and higher than the UUX-Leucine codon usage ratio for rpoS of 0.29 (Table 3). The secondary rationales for this selection were the size of iraP and its involvement in the rpoS regulatory circuitry. The very short ORF of iraP, 258 nucleotides, was ideal for rapid and efficient leu codon engineering. Finally, we reasoned that linking another HULC protein with ms2i6A37 sensitivity would increase the possibility of identifying a physiological relationship between ms2i6A37 and rpoS expression.

TABLE 3.

Leucine codon usage in rpoS and iraP

We constructed an arabinose-inducible iraP258-lacZ translational fusion and a parallel version in which the six UUG and UUA leu codons were changed to CUU codons [P-iraP258(leu*3)-lacZ]. The P-lacZ translation fusions contain the entire iraP ORF except for the stop codon (Fig. 1D). At 15 min, following arabinose induction, the miaA+/miaA− ratio for the P-lacZ fusions is 15:1, and the ratio decreases with time but is still two- to threefold decreased after 30 min (Fig. 1C; Supplemental Table S5). This suggests that MiaA (i6A37 tRNA modification) is necessary for efficient iraP translation. In contrast, the P-iraP258(leu*3)-lacZ was only slightly affected by the absence of MiaA (Fig. 1C). The similar β-galactosidase-specific activity of the miaA+ and miaA P-iraP258(leu*3)-lacZ translational fusions suggests that iraP UUX leu to CXX leu codon mutations suppress the MiaA (i6A37) requirement for iraP translation (Fig. 1C; Supplemental Table S5). Overall these results suggest that UUX leu codons are needed for optimal translation of iraP and changing UUX leu to CUX leu codon mutations decreases the efficiency of iraP translation in the wild-type (miaA+) strain.

Overexpression of leuX (tRNALeuXCAA) suppresses the MiaA requirement for rpoS, but not for rpoS (leu*3) or iraP translation

We hypothesized that overexpression of leucine tRNAs may suppress the effect of the absence of the i6A37 tRNA modification on rpoS translation. If the tRNAs that read UUX leu codons are limiting, leading to the need for the modification for more efficient use, overexpression of these leucine tRNAs may suppress the MiaA requirement for rpoS translation. To test this hypothesis, we measured induction of P-rpoS990-lacZ, in the absence of MiaA, but with overexpression of tRNAs that recognize UUX-Leu codons (Fig. 2). Plasmids carrying either leuX (CAA anticodon) or leuZ (UAA anticodon), under control of an IPTG-inducible promoter, were transformed into rssB− miaA+ and rssB− miaA− P-rpoS990-lacZ translational fusion strains. The activities of these strains were measured at 5-min intervals after addition of arabinose (Fig. 2A,C), or arabinose and IPTG (to induce the tRNAs) (Fig. 2B,D). The leuX-encoding plasmid had very little effect on a wild-type (miaA+) strain, with or without IPTG induction (Fig. 2A,B). However, the leuX-encoding plasmid significantly increased expression of the fusion in the miaA mutant strain (Fig. 2A,B). The increase in expression in the absence of IPTG suggests that there is leaky expression of the tRNALeuXCAA. No increase in the β-galactosidase-specific activity of the miaA− P-rpoS990-lacZ translational fusion was observed in a parallel experiment in which leuZ, encoding tRNALeuZUAA, was overexpressed (Fig. 2C,D). Taken together, this suggests that overexpression of tRNALeuXCAA, but not tRNALeuZUAA, can partially suppress the MiaA requirement for rpoS translation.

FIGURE 2.

The effect of tRNA and tRNA expression on wild-type and miaA P-rpoS990-lacZ translational fusion activity. The rssB− miaA+ (KMT30003) and rssB− miaA− (KMT30011A) P-rpoS990-lacZ translational fusion strains containing plasmids pBR-pLac (KMT30029 and KMT30035, respectively) or pBR-pLac-leuX (KMT30030 and KMT30036) were assayed for β-galactosidase-specific activity following arabinose induction without (A) or with (B) IPTG. Each value represents the mean of at least three replicate experiments; the error bars represent the standard error of the mean (SEM). The rssB− miaA+ (KMT30003) and rssB− miaA− (KMT30011A) P-rpoS990-lacZ translational fusion strains containing plasmids pBR-pLac (KMT30029 and KMT30035, respectively) or pBR-pLac-leuZ (KMT30031 and KMT30037) were assayed for β-galactosidase-specific activity following arabinose induction without (C) or with (D) IPTG. Each value represents the mean of at least three replicate experiments; the error bars represent the standard error of the mean (SEM).

We hypothesized that LeuX suppression of the ms2i6A37 requirement for rpoS translation would not be observed when the MiaA-sensitive leucine codons were removed. To test this hypothesis, we transformed pBR-pLac and pBR-pLac-leuX into rssB− miaA+ and rssB− miaA− genetic backgrounds of this P-rpoS990(leu*3)-lacZ translational fusion and measured the β-galactosidase-specific activity of the resulting strains after arabinose induction (Fig. 3A). We observed no effect of tRNALeuXCAA expression on the β-galactosidase-specific activity of the fusion in the presence or absence of MiaA. This suggests that the tRNALeuXCAA suppression of the MiaA requirement for rpoS translation is through the decoding of UUA's and UUG's leucine codons in the rpoS ORF.

FIGURE 3.

The tRNA suppression assay for the miaA effect on rpoS (leu*3) and iraP. (A) The rssB− miaA+ (KMT33001) and rssB− miaA− (KMT33002) P-rpoS990(leu*3)-lacZ translational fusion strains containing plasmids pBR-pLac (KMT30029 and KMT30035, respectively) or pBR-pLac-leuX (KMT30030 and KMT30036) were assayed for β-galactosidase-specific activity following arabinose and IPTG induction. (B) The miaA+ (KMT45000) and miaA− (KMT45002) P-iraP258-lacZ translational fusion strains containing plasmids pBR-pLac (KMT45003 and KMT45006, respectively) or pBR-pLac-leuX (KMT45004 and KMT45007) were assayed for β-galactosidase-specific activity following arabinose and IPTG induction. (C) The miaA+ (KMT45000) and miaA− (KMT45002) P-iraP258-lacZ translational fusion strains containing plasmids pBR-pLac (KMT45003 and KMT45006, respectively) or pBR-pLac-leuZ (KMT45005 and KMT45008) were assayed for β-galactosidase-specific activity following arabinose and IPTG induction. Each value represents the mean of at least three replicate experiments; the error bars represent the standard error of the mean (SEM).

We carried out similar experiments with the P-iraP258-lacZ translational fusions (Fig. 3B,C). Neither tRNALeuXCAA nor tRNALeuZUAA had a significant effect on the β-galactosidase-specific activity of the P-iraP258-lacZ translational fusion in the absence of MiaA (Fig. 3B,C). This suggests that tRNALeuXCAA is not or is only a minimally effective suppressor of the MiaA requirement for iraP translation. Possible explanations for the difference between rpoS and iraP include the higher number of UUX leu codons in rpoS (13) compared to iraP (six) or the existence of tandem UXX-leu codons in iraP versus the absence of such in rpoS (Tables 1, 2).

TABLE 2.

Silent leucine mutations in the iraP leu* alleles

Mutation of UUA-Leu to CUU-Leu or CUC-Leu codons suppress the MiaA requirement for efficient rpoS translation

To further characterize the role of the ms2i6A37 tRNA modification in the decoding of Leu codons in rpoS and iraP, we constructed translational lacZ fusions to alleles of rpoS and iraP where either UUA- or UUG-Leucine codons were removed and replaced with either a CUC or CUU leucine codon. We designate the UUA-Leu to CUU-Leu or CUC-Leu rpoS alleles as leu*1 alleles. We designate the UUG-Leu to CUC-Leu rpoS alleles as leu*2 alleles. The precise amino acid number and mutation are listed in Table 1. We then tested the β-galactosidase-specific activity of these fusions as described in the Materials and Methods section. We also repeated the assay in Figure 1A, with wild-type and leu*3 rpoS alleles, as a control for the leu*1 and leu*2 rpoS alleles (Fig. 4A). In the presence of MiaA, the rpoS leu*1 allele has a modest decrease in β-galactosidase-specific activity between 10 and 25 min following induction (Fig. 4B). The miaA+ ratio of wild type to leu*1 is increased by 1.2 to 1.5-fold at or beyond 15 min of induction (Supplemental Table S4). This suggests that the UUA-Leu to CXX-Leu silent codon mutations slightly decrease efficiency of rpoS translation. In the absence of MiaA, at or beyond 15 min of induction, the β-galactosidase-specific activity of the P-rpoS990(leu*1)-lacZ allele is at least twofold greater than the P-rpoS990-lacZ allele. The activities of the miaA+ and miaA− P-rpoS990(leu*1)-lacZ translational fusion are relatively similar in comparison to the miaA+ and miaA− P-rpoS990-lacZ translational fusion (Fig. 4B). The miaA+/miaA− ratio of the P-rpoS990(leu*1)-lacZ and P-rpoS990-lacZ translational fusions, 15 min following induction, are 1.8 and 23, respectively. This ratio is much closer to 1.0-fold increase in the leu*1 fusion as opposed to the wild-type fusion (Supplemental Table S3). This suggests that MiaA and the ms2i6A37 tRNA modification are necessary for efficient translation of UUA-Leucine codons within the rpoS ORF.

FIGURE 4.

Translation of rpoS with UUG- or UUA-Leucine codons replaced with CXX-Leucine codons. (A) The rssB− miaA+ (KMT33001) and rssB− miaA− (KMT33002) P-rpoS990(leu*3)-lacZ as well as rssB− miaA+ (KMT30003) and rssB− miaA− (KMT30011A) P-rpoS990-lacZ translational fusion strains were assayed for β-galactosidase-specific activity following arabinose induction. (B) The rssB− miaA+ (KMT) and rssB− miaA− (KMT) P-rpoS990(leu*2)-lacZ as well as rssB− miaA+ (KMT30003) and rssB− miaA− (KMT30011A) P-rpoS990-lacZ translational fusion strains were assayed for β-galactosidase-specific activity following arabinose induction. (C) The rssB− miaA+ (KMT37002) and rssB− miaA− (KMT37003) P-rpoS990(leu*2)-lacZ as well as rssB− miaA+ (KMT30003) and rssB− miaA− (KMT30011A) P-rpoS990-lacZ translational fusion strains were assayed for β-galactosidase-specific activity following arabinose induction. (D) The rssB− miaA+ (KMT36002) and rssB− miaA− (KMT36003) P-rpoS990(leu*1)-lacZ, rssB− miaA+ (KMT37002) and rssB− miaA− (KMT37003) P-rpoS990(leu*2)-lacZ, rssB− miaA+ (KMT33001) and rssB− miaA− (KMT33002) P-rpoS990(leu*3)-lacZ as well as rssB− miaA+ (KMT30003) and rssB− miaA− (KMT30011A) P-rpoS990-lacZ wild-type translational fusion strains were assayed for β-galactosidase-specific activity following 30 min of arabinose induction. Each value represents the mean of at least three replicate experiments; the error bars represent the standard error of the mean (SEM). The means between miaA+ and miaA− strains in section D were analyzed for statistical significance using t-test and the Holm–Sidak method with P-values <0.05.

UUG-Leu to CXX-Leu codon mutations decrease rpoS translational efficiency

In the presence of MiaA, the leu*2 alleles are severely defective for translational β-galactosidase-specific activity, decreased by approximately fourfold starting at 10 min after induction (Fig. 4C; Supplemental Table S4). This suggests that, independent of MiaA and the ms2i6A37 tRNA modification, the presence of UUG-Leucine codons is critical for efficient translation of the rpoS ORF. Furthermore, the β-galactosidase-specific activity of the miaA+ P-rpoS990(leu*2)-lacZ translational fusion is nearly identical to the miaA− P-rpoS990(leu*2)-lacZ translational fusion (Fig. 4; Supplemental Table S3). However, the severely compromised rpoS translation seen in the absence of UUG possibly makes this explanation too simple. Interestingly, there is no statistically significant difference, at the 30-min time point following induction, between miaA+ and miaA− activities of the leu*1 and leu*2 alleles of the P-rpoS990-lacZ translational fusion (Fig. 4D). In contrast, there is a statistically significant difference, at the 30-min time point following induction, between miaA+ and miaA− activities of the wild-type and leu*3 alleles of the P-rpoS990-lacZ translational fusion (Fig. 4D). The removal of UUA-Leu only or UUG-Leu codons only, as opposed to the removal of both, results in stronger suppression of the MiaA requirement during rpoS expression. The reason for this is unclear.

UUA-Leu to CUU-Leu or UUG-Leu to CUU-Leu codon mutations modulate iraP translational efficiency and partially suppress the MiaA requirement during iraP translation

While it is clear from data in Figure 1C that removal of both UUA-Leucine and UUG leu codons within iraP suppresses the MiaA effect during iraP translation, we sought to further characterize and define the relative contributions of UUA leu and UUG leu to the MiaA requirement for iraP translation just as we did for rpoS translation. We created two additional alleles of the P-iraP258-lacZ translational fusion, P-iraP258(leu*1)-lacZ and P-iraP258(leu*2)-lacZ. P-iraP258(leu*1)-lacZ consists of UUA-Leucine to CUU-Leucine codon changes alone (Table 2). P-iraP258(leu*2)-lacZ consists of UUG-Leucine to CUU-Leucine codon changes alone (Table 2). We measured the β-galactosidase-specific activity of these fusions as described in the Experimental Design section of Materials and Methods. Upon comparison of the miaA+ P-iraP258(leu*1)-lacZ and miaA+ P-iraP258-lacZ translational fusion β-galactosidase-specific activity after induction, it is clear that the leu*1 mutation increases iraP translational efficiency (Fig. 5B), most evident at 15–30 min following induction (Fig. 5B). The miaA+ P-iraP258-lacZ/P-iraP258(leu*1)-lacZ ratio is between −1.6 to −1.7 during this time period, demonstrating an increase in iraP translational efficiency in the absence of UUA-Leucine codons (Supplemental Table S6). The β-galactosidase-specific activity of the miaA− P-iraP258(leu*1)-lacZ fusion is 2.1-fold less than the miaA+ P-iraP258(leu*1)-lacZ, after 30 min of induction (Fig. 5B; Supplemental Table S5). This is slightly smaller than the 2.8-fold decrease seen after 30 min (Supplemental Table S5). The miaA+/miaA− β-galactosidase-specific activity ratio, 15 min after induction, was 1.8 in the leu*1 allele versus 15 in the wild-type (non leu*) allele of the P-iraP258-lacZ translational fusion (Supplemental Table S5). This suggests that the UUA-Leucine to CUU-Leucine codon changes lead to partial suppression of the MiaA requirement for iraP translation.

FIGURE 5.

Translation of iraP with UUG- or UUA-Leucine codons replaced with CXX-Leucine codons. (A) The miaA+ (KMT45000) and miaA− (KMT45002) P-iraP258(leu*3)-lacZ as well as miaA+ (KMT42000) and miaA− (KMT42002) P-258-lacZ translational fusion strains were assayed for β-galactosidase-specific activity following arabinose induction. (B) The miaA+ (KMT43000) and miaA− (KMT43002) P-iraP258(leu*1)-lacZ as well as miaA+ (KMT42000) and miaA− (KMT42002) P-iraP258-lacZ translational fusion strains were assayed for β-galactosidase-specific activity following arabinose induction. (C) The miaA+ (KMT44000) and miaA− (KMT44002) P-iraP258(leu*2)-lacZ as well as miaA+ (KMT42000) and miaA− (KMT42002) P-iraP258-lacZ translational fusion strains were assayed for β-galactosidase-specific activity following arabinose induction. (D) The miaA+ (KMT43000) and miaA− (KMT43002) P-iraP258(leu*1)-lacZ, the miaA+ (KMT44000) and miaA− (KMT44002) P-iraP258(leu*2)-lacZ, the rssB− miaA+ (KMT45000) and rssB− miaA− (KMT45002) P-iraP258(leu*3)-lacZ as well as miaA+ (KMT42000) and rssB− miaA− (KMT42002) P-iraP258-lacZ wild-type translational fusion strains were assayed for β-galactosidase-specific activity following 30 min of arabinose induction. Each value represents the mean of at least three replicate experiments; the error bars represent the standard error of the mean (SEM). The means between miaA+ and miaA− strains in section D were analyzed for statistical significance using t-test and the Holm–Sidak method with P-values <0.05.

We measured the β-galactosidase-specific activity of the P-iraP258(leu*2)-lacZ translational fusion after induction, in the presence and absence of MiaA to determine the relative contribution of UUG-Leucine codons to iraP translation (Fig. 5; Supplemental Table S6). UUG-Leucine to CUU-Leucine codons had no effect on the translation of iraP in a miaA+ host (Fig. 5C; Supplemental Table S6). However, the β-galactosidase-specific activity of the miaA− P-iraP258(leu*2)-lacZ translational fusion is higher than the miaA− P-iraP258(leu*2)-lacZ (Fig. 5C). Furthermore, the miaA+/miaA− ratios in the leu*2 allele of the iraP fusion are less than the miaA+/miaA− ratios in the wild-type allele of the iraP fusion (Fig. 5C,D; Supplemental Table S5). Taken together this suggests that the leu*2 allele allows for suppression of the MiaA requirement for iraP translation. More precisely, MiaA is necessary for iraP translation in part due to decoding of UUG-Leucine codons.

MiaB is dispensible for rpoS and iraP translation

As previously discussed, the MiaA enzyme catalyzes the first step in the synthesis of the ms2i6A37, the addition of the i6 to the A37 nucleotide (Bartz et al. 1970). The addition of the ms2 group, catalyzed by the MiaB enzyme, requires the presence of the i6 as a prerequisite (Vold et al. 1979). Although we had previously demonstrated no effect of miaB mutants on rpoS levels (Thompson and Gottesman 2014), we revisited this question using the rpoS and iraP translational fusions. We measured the β-galactosidase-specific activity of the P-rpoS990-lacZ and P-iraP258-lacZ translational, following arabinose induction (Supplemental Fig. S9). For the rpoS translational fusion, there was a negligible decrease in the β-galactosidase-specific activity of the fusion in the miaB mutant, at 15–30 min following arabinose induction. For the iraP translational fusion, there was a negligible decrease in the β-galactosidase-specific activity of the fusion in the miaB mutant, at 20–30 min following arabinose induction. Taken together, this confirms our previous finding, and shows that the ms2 portion of the ms2i6A37 tRNA modification is dispensable for full rpoS and iraP translation. The i6A37 portion of the ms2i6A37 tRNA modification is responsible for its role in translation of rpoS and iraP.

DISCUSSION

Prediction of i6A37 modification tunable transcripts (MoTTs) through leucine codon usage

Endres et al. (2015) define transcripts with codon usage different from that of average transcripts, as modification tunable transcripts (MoTTs). In their model, under normal growth conditions, MoTTs are moderately expressed in the presence of tRNA modifications (Endres et al. 2015). Levels of tRNA modification, and therefore the translation of MoTTs, change during stress or damage conditions (Endres et al. 2015). Our findings for rpoS and iraP establish them as MoTTs, adding MiaA modification and UUX codon usage as new examples of this phenomenon.

We hypothesized that UUX-Leucine codon enrichment within an ORF may correlate with MiaA (i6A37) sensitivity in genes other than rpoS (Thompson and Gottesman 2014). The data in this study strongly support the utility of using leucine codon usage ratios to predict the necessity of the i6A37 tRNA modification during translation of a given protein and provides a possible method for the identification of this subclass of MoTTs as HULC genes. We observed that the ratio of UUX-Leu codons in iraP is 0.46, which is even greater than the 0.29 ratio seen in rpoS (Table 3). While both ratios are higher than the expected 0.22, the ratio is much higher in iraP. Further bioinformatics searches, along with biochemical and genetic analysis, will be needed to further evaluate the predictive power associated with the correlation of UUX-Leu codon usage and sensitivity to the lack of MiaA for putative HULC proteins. These investigations are likely to yield more insight into the regulatory nature of the i6A37 tRNA modification in E. coli and other biological systems.

UUG-Leucine and UUA-Leucine codon usage in rpoS and iraP

There are differences in how UUA and UUG codons are used in rpoS and iraP, in relation to the presence of the i6A37 modification. Our leu* mutations were created with the idea that UUX-Leucine to CUX-Leucine mutations would suppress the MiaA requirement for full rpoS translation. The leu*3 mutation suppressed the MiaA effect in both rpoS and iraP (Figs. 4, 5). However, the suppression was more pronounced in iraP, as miaA+ and miaA− levels were identical (Fig. 5). The reason for this is unclear, particularly since iraP has seven fewer UUX-Leu codons than rpoS. Both, leu*1 and leu*2 mutations, representing the UUA-CUX and UUG-CUX substitution mutations, respectively, demonstrated stronger MiaA suppression than the leu*3 mutation for reasons that are not clear. However, this suggests that UUA-Leu and UUG-Leu substitutions, individually, are more effective at suppressing the MiaA effect than UUA and UUG substitutions collectively. It should be noted that the rpoS transcript is approximately three times longer than the iraP transcript. UUG-Leucine codons are more frequent within the rpoS and iraP transcripts than UUA-Leucine codons. The number of UUX codons in iraP and rpoS transcripts is seven and 12, respectively. There are two instances within the iraP transcript where UUX codons occur in tandem, once at leucine 8 and leucine 9 and again at leucine 80 and leucine 81. MiaA affects the translation of the 14 amino acid trp leader region, trpL, by influencing the translation rate of two tandem Trp codons (Trp 10 and Trp11) by ms2i6A37-modified tRNATrp (Landick et al. 1990). This tandem duplication of UUX-Leucine codons is not seen in the rpoS transcript. These two independent tandem duplications of UUX-Leucine codons in the iraP ORF likely contribute significantly to MiaA sensitivity during iraP translation. Undermodified tRNALeu may contribute to ribosome pausing or stalling at each of these two tandem duplications contributing to decreased translational speed and accuracy.

The steady-state levels of rpoS have an additional level of regulatory fine-tuning

rpoS is subject to regulation at multiple levels, including the translation of the rpoS ORF, through SsrA, MiaA, AceE, and the presence of rare codons (Ranquet and Gottesman 2007; Kolmsee and Hengge 2011; Thompson and Gottesman 2014; Battesti et al. 2015). While there have been extensive studies on the regulation of the synthesis and activity of tRNA modification enzymes, the precise cellular or environmental signals that influence the post-transcriptional levels and post-synthesis activities of many tRNA modifications are elusive (Winkler 1998). This is also true of the i6A37 modification and the activity of MiaA in E. coli. The leucine codon usage relationship suggests that leucine availability may provide a physiological condition whereby the i6A37 modification is most critical for expression of rpoS synthesis. Lrp, the leucine-responsive regulatory protein, is a global regulatory protein whose regulatory action is partially dependent upon leucine (Ernsting et al. 1992; Lin et al. 1992; Platko and Calvo 1993). Mutations in lrp, as well as rpoS, confer a growth advantage during stationary phase, or GASP, phenotype (Zambrano et al. 1993; Zinser and Kolter 2000). In addition, the transcription factor LeuO regulates DsrA, one of the small RNAs that stimulate rpoS translation (Klauck et al. 1997; Repoila and Gottesman 2001). LeuO also regulates the leucine biosynthesis operon, leuABCD (Chen et al. 2001; Stratmann et al. 2012). These observations provide clues into a stationary phase network that connects leucine metabolism and rpoS synthesis.

There are several environmental signals that feed into the rpoS synthesis pathway through the small regulatory RNAs that stimulate its translation and anti-adaptors that promote its protein stability. Low temperature (DsrA), cell surface stress (RprA), aerobiosis (ArcZ), phosphate starvation (iraP), magnesium starvation (IraM), and DNA Damage (IraD) all act on RpoS synthesis or stability (Sledjeski et al. 1996; Majdalani et al. 2002; Bougdour et al. 2006, 2008; Mandin and Gottesman 2010). It would be useful to consider the possibility, and investigate, whether ms2i6A37 modification levels are modulated under these conditions that are known to stimulate Rpos levels. This may lead to greater insight into the physiological and regulatory nature of the ms2i6A37 tRNA modification.

MiaA is encoded directly upstream of and co-transcribed with Hfq, which is necessary for rpoS translation mainly through acting as a chaperone for the activating sRNAs (Tsui and Winkler 1994; Tsui et al. 1994). This tandem coding of MiaA and Hfq is highly conserved throughout the prokaryotic domain. This and our previous work offer one possible explanation, the regulation of rpoS translation, for this conserved synteny of miaA and hfq. It is likely that further bioinformatics and experimental analysis will uncover other mRNAs whose translation is regulated by both Hfq (possibly through Hfq-dependent sRNAs) as well as the i6A37 tRNA modification.

There is some residual β-galactosidase-specific activity of the rpoS and iraP leu* fusions, in which all UUX leu codons have been changed to CUX codons, in the absence of miaA (Figs. 1, 4, 5). This suggests that MiaA may be necessary for the decoding of other (non-leu) codons within rpoS and iraP, or have some other indirect effects on rpoS and iraP translation. We previously identified tsaE as a gene that now falls into the HULC designation of proteins and consequently may be an i6A37 MoTT (Thompson and Gottesman 2014). The TsaE protein is also encoded upstream of miaA and hfq (Tsui and Winkler 1994). The TsaE protein, along with TsaC, TsaD, and TsaB, form an enzyme that catalyzes the addition of the N6-L-threonylcarbamoyladenine37 (t6A37) modification on ANN decoding tRNAs (Deutsch et al. 2012; Zhang et al. 2015). It is possible that translation of both rpoS and iraP are sensitive to loss of the t6A37 modification, and that i6A37 deficiency affects rpoS and iraP levels in part indirectly by limiting TsaE translation. We are currently investigating the possible role of t6A on rpoS and iraP levels to test this hypothesis.

Implications for comparative functional genomics of tRNA isopentenyl transferases

We previously noted other proteins within the prokaryotic domain that are sensitive to MiaA levels (Thompson and Gottesman 2014). These proteins include Agrobacterium tumefaciens vir and Shigella flexneri VirF (Gray et al. 1992; Durand et al. 1997, 2000). In addition, Streptomyces coelicolor bld mutant phenotype is suppressed by overexpression of tRNACAALeu, which also likely contains the i6A37 modification (Pettersson and Kirsebom 2011). The UUX-Leucine codon usage in Shigella flexneri VirF is indicative of MiaA sensitivity (Thompson and Gottesman 2014). Based on our observations in E. coli K12, it is likely that experimental analysis of leucine codon usage within these genes will yield insights into how UUX-Leu codons influence the translation of MiaA-sensitive proteins in other bacterial species.

The MiaA gene is also highly conserved among other biological domains, with homologs in yeast and humans, MOD5 and TRIT1, respectively (Martin and Hopper 1982; Golovko et al. 2000; Lamichhane et al. 2013b; Yarham et al. 2014). TRIT1 is particularly interesting due to its role as a putative lung adenocarcinoma tumor suppressor, as its expression was significantly down regulated in lung adenocarcinomas (Spinola et al. 2005). A genome-wide association study of a single nucleotide polymorphism (SNP) mapping to TRIT1, in different ethnic populations, demonstrated statistically significant links between a TRIT1 Phe202Leu genotype and lung cancer survival rates (Spinola et al. 2007). This suggests that an association of leucine codon usage and tRNA isopentenyl transferase activity may be highly conserved well beyond the bacterial domain.

Expansion of the rpoS regulatory network in E. coli

Prior to this work, the mechanism by which MiaA exerted its effect on rpoS expression was undefined. Our data strongly suggest that the MiaA requirement for rpoS expression is partially through promotion of translational efficiency at MiaA-sensitive UUA- and UUG-Leucine codons in the rpoS ORF. The rpoS ORF is somewhat enriched for these leucine codons in comparison to other proteins that are gene regulators or members of the transcriptional regulatory machinery (Thompson and Gottesman 2014). Our current model for the role of MiaA in rpoS regulation is illustrated in Figure 6. In the presence of active, or excess MiaA, i6A37-modified tRNALeuCAA efficiently recognizes UUA or UUG codons within the rpoS and iraP ORFs, leading to optimal translation of rpoS and iraP ORFs (Fig. 6). iraP levels contribute to rpoS stability, further increasing steady-state levels of rpoS (Fig. 6). In the absence of MiaA, or the presence of inactive MiaA, unmodified or undermodified tRNALeuCAA leads to the suboptimal translation of rpoS and iraP ORFs (Fig. 6). Suboptimal iraP levels lead to decreased rpoS stability, further decreasing steady-state levels of rpoS (Fig. 6).

FIGURE 6.

MiaA (i6A37) regulation of rpoS and iraP model. This model illustrates our current model for the i6A37 tuning of rpoS and iraP expression. In the presence of excess and/or active MiaA, tRNA is i6A37 modified, leading to optimal translation of UXX-Leucine codons with both rpoS and iraP. Since iraP increases rpoS stability, optimal amounts of iraP contribute to increased steady-state levels of rpoS. When MiaA is inactive or limiting, tRNA is unmodified, leading to suboptimal translation of UXX-Leucine codons within both rpoS and iraP. Suboptimal translation of iraP will result in decreased stability of rpoS. These combined factors significantly decrease the steady-state levels of rpoS.

MATERIALS AND METHODS

Media and growth conditions

M63-Glycerol-Sucrose-XG plates were used for positive selection of in-frame and out-of-frame P-rpoS990-lacZ translational fusion (Court et al. 2003; Mandin and Gottesman 2010). Positive selection of in-frame and out-of-frame P-rpoS990-lacZ translational fusions were confirmed by screening sucrose-resistant (SucR) colonies for chloramphenicol-sensitivity (CmS) on Luria-Bertani (LB) Lennox Agar plates supplemented with chloramphenicol to a final concentration of 25 µg/mL. LB agar plates supplemented with zeomycin (LB-Zeo) or tetracycline (LB-Tet), to a final concentration of 25 µg/mL, were used to select for transduction or recombineering of rssB::tet or ΔmiaA::zeo mutations, respectively. LB agar plates supplemented with ampicillin (LB-amp), to a final concentration of 100 µg/mL were used for selection of plasmids. All cultures for β-galactosidase assays were grown in LB Lennox media (KD Medical). LB Lennox media were supplemented with ampicillin to a final concentration of 100 µg/mL selection of pBR-leuX or pBR-leuZ plasmids. For arabinose induction experiments, cells were grown in LB Lennox containing 0.2% glucose (LB-Glu) to an OD600 of 1.0, washed with an equal volume of fresh LB Lennox to remove residual glucose, and resuspended in LB Lennox with 0.2% arabinose. Upon shifting cultures to LB-Ara, aliquots of the cultures were taken periodically following arabinose induction for β-galactosidase assays.

Strains and plasmids

Strains and plasmids are listed in Supplemental Table S1. All P-rpoS990-lacZ translational fusion strains used for the experiments carried a mutation in rssB, rssB::tet, to rule out possible effects on rpoS stability.

Construction of P-rpoS990-lacZ translational fusions

Arabinose-inducible translational fusions of rpoS990 to lacZ were constructed by recombineering strain PM1805. Strain PM1805 contains the λ-Red proteins under control of the temperature-sensitive allele of the λ-repressor, cI857. PM1805 also contains the counter-selectable marker, cat-sacB, at the lac locus to allow for positive selection of recombinant fusions in the presence of sucrose. Recombineering into PM1805 requires induction of the λ-Red proteins, creation of electrocompetent cells, electroporation of allelic exchange substrates, and positive selection of fusions in the presence of sucrose. To induce the λ-Red proteins, PM1805 was grown in LB at 32°C to OD600 of 0.5 and shifted to 43.5°C for 15 min. The culture was then cooled in an ice-water bath. Then, washing the induced PM1805 culture in ice-cold H2O made electrocompetent cells. Approximately 100 ng of the purified PCR product corresponding to the allelic exchange substrates were electroporated into the electrocompetent-induced PM1805 using the GenePulser (BioRad) on the Ec1 setting. Electroporated cells were recovered overnight in 10 mL of LB (Lennox) broth in a 125 mL flask at 32°C. Overnight cultures were serially diluted and spread on M63-Glycerol-Sucrose-XG plates and incubated at 30°C for 3–5 d. The purified PCR products were electroporated into electrocompetent PM1805. For construction of the wild-type P-rpoS990-lacZ translation fusion, an allelic exchange substrate was created through the amplification of a portion of the P-rpoS990-lacZ translational fusion from genomic DNA of strain KMT581 using oligonucleotide primers KT1123 and KT1124 (Table 2).

Three different derivatives of the P-rpoS990-lacZ translation fusion were also constructed: P-rpoS990(leu*1)-lacZ, P-rpoS990(leu*2)-lacZ, P-rpoS990(leu*3)-lacZ translational fusions. The P-rpoS990(leu*1)-lacZ contained UUA to CUU changes across the entire ORF. The P-rpoS990(leu*2)-lacZ contained UUG to CUC changes across the entire ORF. The P-rpoS990(leu*3)-lacZ contained both UUA to CUU and UUG to CUC changes across the entire ORF, effectively removing all MiaA-sensitive leucine codons. For the P-rpoS990(leu*1)-lacZ P-rpoS990(leu*2)-lacZ translational fusions, synthetic (gBlock) DNA fragments (IDT Technologies) “P-rpoS990(leu*1)-lacZ_AES” and “P-rpoS990(leu*2)-lacZ_AES” (Supplemental Table S2), respectively, were PCR amplified using oligonucleotide primers KT1123 and KT1124 and used as an AES for recombineering into electrocompetent PM1800. For the P-rpoS990(leu*3)-lacZ translational fusions, two synthetic (gBlock) DNA fragments (IDT Technologies) with overlapping homology, “P-rpoS990(MiaA_Leu-) gBlockup” and “P-rpoS990(MiaA_Leu-) gBlockdown” (Supplemental Table S2), were recombined together using the Gibson Assembly Mastermix (New England Biolabs). The resulting DNA fragment was PCR amplified using oligonucleotide primers KT1123 and KT1124 (Table 2) and used as an AES for recombineering into electrocompetent PM1805.

Construction of ΔmiaA::zeo allele by recombineering

The miaA gene was deleted and replaced with a zeomycin (zeo) resistance cassette by recombineering in strain KMT194. An allelic exchange substrate for miaA mutagenesis was created by PCR amplification of the zeo cassette from genomic DNA from strain KMT465, using oligonucleotide primers KT1035 and KT1036 (Table 2). The PCR product was purified and ∼100 ng was used for electroporation into electrocompetent KMT194 after induction of the λ-Red proteins, using a protocol identical to the one used to induce the λ-Red proteins in, and prepare electrocompetent cells of, PM1805. After electroporation, putative recombinants carrying ΔmiaA::zeo mutations were selected on LB-Zeo plates. Recombinants were purified once on LB-Zeo plates and twice on LB plates. The ΔmiaA::zeo mutation was confirmed by PCR and phenotypic analysis.

Construction of P-iraP258-lacZ translational fusions

We constructed arabinose-inducible translational fusions of the entire iraP ORF, except for the termination codon iraP258, to lacZ by recombineering strain PM1805. The methods used were identical to those listed in the construction of the P translational fusion strains. We also created three different derivatives of the P-iraP258-lacZ translation fusion in a manner identical to the rpoS fusions: P-iraP258(leu*1)-lacZ, P-iraP258(leu*2)-lacZ, P-iraP258(leu*3)-lacZ translational fusions. The P-iraP258(leu*1)-lacZ contained UUA to CUU changes across the entire ORF. The P-iraP258(leu*2)-lacZ contained UUG to CUU changes across the entire ORF. The P-iraP258(leu*3)-lacZ translational fusion contained both UUA to CUU and UUG to CUU changes across the entire iraP ORF, effectively removing all MiaA-sensitive leucine codons from the iraP portion of the fusion. For the P-iraP258-lacZ, P-iraP258(leu*1)-lacZ, P-rpoS990(leu*2)-lacZ, and P-iraP258(leu*3)-lacZ translational fusions, synthetic (gBlock) DNA fragments (IDT Technologies) “P- iraP258-lacZ_AES,” “P-iraP258(leu*1)-lacZ_AES,” “P-rpoS990(leu*2)-lacZ_AES,” and “P-iraP258(leu*3)-lacZ AES” (Supplemental Table S2), respectively, were PCR amplified using oligonucleotide primers KT1162 and KT1163 and used as an AES for recombineering into electrocompetent PM1800.

P1 transduction to moved marked mutations

All ΔrssB::tet and ΔmiaA::zeo mutations were transferred into the wild-type and mutant P-rpoS990-lacZ or P-iraP258-lacZ translational fusion strains by bacteriophage P1 transduction and selecting for tetracycline (TetR) or zeomycin (ZeoR) resistance, respectively.

DNA manipulations and cloning reactions

To determine whether overexpression of rare leucine tRNAs could suppress the MiaA requirement for rpoS expression, we cloned rare leucine tRNA genes, leuX and leuZ, downstream from the IPTG-inducible PLlacO promoter in plasmid pBR-pLac (Guillier and Gottesman 2006).

Construction of pBR-leuX

The leuX gene was amplified from E. coli K12 MG1655 chromosomal DNA using oligonucleotide primers KT1104 and KT1105 (Table 2). The PCR product was then purified using the PCR Purification Kit (Lamda Biotech), digested with restriction enzymes AatII (New England Biolabs) and EcoRI (New England Biolabs), and ligated to an AatII/EcoRI digest of plasmid pBR-pLac (Supplemental Table S1) using Quick Ligase (New England Biolabs). An aliquot of the ligation reaction was transformed into NEB5 chemically competent cells (New England Biolabs) using heat shock transformation at 42°C for 30 sec, recovered in 500 µL SOC Media, and 100 µL aliquot was spread on LB-Amp plates. Putative pBR-leuX transformants were screened by colony PCR using the BAC-Direct Kit and oligonucleotide primers KT1115 and KT1105. Plasmids from PCR positive transformants were sequenced to confirm the presence of the leuX insert. Plasmid pBR-leuX was transformed into wild-type and ΔmiaA::zeo fusion strain using TSS transformation (Chung et al. 1989). The clones were confirmed by DNA sequencing.

Construction of pBR-leuZ

The steps used for construction of pBR-leuZ are identical to those used for the construction of pBR-leuX with the exception of the oligonucleotide primers used for PCR amplification (oligonucleotide primers KT1102 and KT1103) and colony PCR screening (oligonucleotide primers KT1115 and KT1103). The clones were confirmed by DNA sequencing.

β-galactosidase assays

High-throughput kinetic β-galactosidase assays were carried out in 96-well plates as previously described (Zhou and Gottesman 1998). The Filtermax F5 (Molecular Devices) multimode microplate reader was used to read microtiter plates. β-galactosidase-specific activity units are defined as the slope of OD420 reading divided by OD600 and are approximately 25-fold lower than Miller Units.

Experimental design for assays executed following arabinose induction

Briefly, samples to be assayed were grown in 5 mL of LB-Glu overnight at 37°C in a roller drum. Overnight cultures were diluted 1:1000 in 30 mL of fresh LB-Glu in a 125–mL Erlenmeyer flask and grown at 37°C in a shaking water bath at 200 rpm. When cultures reached an OD600 of 1.0, cells were harvested by centrifugation and resuspended in 30 mL of fresh LB supplemented 0.2% arabinose, and 100 µL aliquots of each culture were taken every 5 min for β-galactosidase assays. Samples were collected in triplicate for each individual experiment and averages were taken as a representative sample for each experiment. The data represent the mean and standard error of the mean of at least three independent replicates.

SUPPLEMENTAL MATERIAL

Supplemental material is available for this article.