To determine the effect of mG37 deficiency on the biosynthesis of membrane proteins, we used quantitative proteomics to measure protein levels in the membrane fraction of E. coli trmD-KD cells grown with or without Ara. A total of 226 membrane proteins, 47 of which were associated with the OM, were analyzed by label-free quantification to determine fold-changes between Ara+ and Ara− conditions. While non-OM proteins were on average up-regulated in the absence of Ara by 16% (median increase of 2= 1.16), OM proteins were on average down-regulated by 21% (median decrease of 2= 0.79) ( Figure 2 A). Of interest were LolB and OmpA, responsible for stable anchoring of drug-efflux pumps to the OM () and for anchoring the OM to the peptidoglycan cell wall, respectively. lolB and ompA are enriched with Pro codons relative to the average codon usage in E. coli protein-coding genes ( Figure 2 B, lolB: CCN [6.7% versus 4.3%] and CC[C/U] [2.4% versus 1.1%]) and ompA CCN [5.5% versus 4.3%]). This enrichment is specific because their usage of Leu codons (CUN), which also require mG37 for translation, is typical ( Figure S3 D). The enrichment of Pro codons in lolB and ompA supports the notion that their decrease in protein levels is correlated with the poor translation of Pro codons in mG37-deficient cells. Western blot analysis showed that the amount of LolB relative to the cytosolic cysteinyl-tRNA synthetase CysRS () in mG37-deficient cells decreased to 26% in E. coli and to 56% in Salmonella ( Figures 2 C and S3 E), while relative mRNA levels were unaffected ( Figure S3 F), indicating that the reduction in protein levels was due to reduced translation. These data are consistent with the notion that translation of lolB involves a TrmD-dependent codon at the 2and 4positions of the E. coli gene and at the 4position of the Salmonella gene ( Figure 1 A), whereas translation of cysS (for CysRS) involves no such codons in the first 16 positions. Western blot analysis also showed that the amount of OmpA relative to CysRS decreased to 72% in mG37-deficient E. coli cells ( Figure 2 D), providing additional support for the notion that translation of membrane-associated genes that are enriched with Pro codons is sensitive to the loss of mG37.

(C and D) m 1 G37 deficiency decreased LolB levels (C) to 26% in E. coli and to 56% in Salmonella, and decreased OmpA levels in E. coli to 72% (D) in western blots (top). Overnight cultures of trmD-KD cells were diluted 1:100 into fresh LB with or without 0.2% Ara and grown for 4 h at 37°C. Cells were inoculated into fresh LB in Ara+/− conditions for another 3 h. Data and error bars represent mean ± SD, n = 4.

(B) Frequency of Pro codons CCN (top) and CC[C/U] (bottom) in genes whose OM proteins are reduced in Ara– versus Ara+ in (A). Each frequency is compared to the average frequency of respective Pro codons in E. coli protein-coding genes.

(A) Quantitative mass spectrometry analysis of membrane proteins in E. coli trmD-KD cells isolated from Ara− and Ara+ conditions. The label-free quantification intensity is compared to the signal of log 2 (fold-change) (Ara–/Ara+). OM proteins are plotted in black with a vertical line indicating the median of −0.33 (equivalent to a decrease of 21%), while non-OM proteins are plotted in blue with a vertical line showing the median of 0.22 (equivalent to an increase of 16%). p < 0.001 by a Kolmogorov-Smirnov analysis.

Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

To determine how mG37 controls codon-specific translation of membrane-associated genes, we created trmD-KD (knockdown) strains of E. coli and Salmonella. Since trmD is essential for cell viability () and cannot be deleted, we created each trmD-KD strain by deleting the chromosomal trmD ( Figures S2 A and S2B) while expressing the human counterpart trm5 from a plasmid with an arabinose (Ara)-inducible promoter. We previously showed that Trm5 is capable of supplying mG37-tRNA to support bacterial viability () but that it is unstable in bacteria and can be removed rapidly (). In the E. coli and Salmonella trmD-KD strains, the level of human Trm5 upon Ara induction increased with time and reached a steady state in 1–2 h but decreased rapidly within 30 min upon Ara removal ( Figure 1 D). Cells with Trm5-produced mG37 formed colonies up to a 10-fold dilution, whereas mG37-deficient cells were not viable even without dilution ( Figure 1 E). To determine intracellular mG37 levels, cells were grown with 0.2% Ara to saturation and diluted 1:100 into fresh Luria broth (LB) with or without Ara for 4 h, followed by another dilution to OD= 0.1 in fresh LB with or without Ara and grown for 3 h. These serial passages were necessary to deplete cells of pre-existing mG37-tRNA ( Figure S3 A). Primer extension analysis validated that the UGG isoacceptor of tRNAin trmD-KD cells contained mG37 at 70% and 12% in cultures with and without Ara, respectively ( Figure 1 F). This pattern was preserved for the GGG isoacceptor ( Figures S3 B and S3C) and was consistent with quantitative mass spectrometry analyses of the UGG isoacceptor ( Figure 1 G).

We previously showed that mG37 has the strongest effect on codon-specific translation of CC[C/U] at the 2codon position of an open reading frame and that this effect gradually decreases over the next 15 codons (). In an analysis of the E. coli MG1655 genome, we found that the occurrence of CC[C/U] at the 2codon position is 2-fold higher for genes encoding membrane-associated proteins relative to non-membrane-associated proteins (1.8% versus 0.8%, n = 4,289, p < 0.05, Fisher’s exact test with Bonferroni correction) (). This enrichment was also observed when considering both the 2and 3codon positions (3.7% versus 1.5%, n = 4,289, p < 0.0005, Fisher’s exact test with Bonferroni correction). The over-representation of CC[C/U] is also evident in the genome of Salmonella LT2 (). Among genes with CC[C/U] at the 2codon position, 31% and 26% encode membrane-associated proteins in E. coli and Salmonella, respectively ( Figures 1 C and S1 ). The high prevalence of Pro near the N terminus of membrane proteins is consistent with its role in creating turns of transmembrane domains that cross a lipid bilayer ().

m1G37 Deficiency Causes Membrane Damage and Reduces OM Stiffness

1G37-deficient cells would damage membrane structural integrity. We observed increased intracellular accumulation in m1G37-deficient bacteria of both the redox sensor AlamarBlue, which becomes fluorescent inside cells, and the DNA fluorescent stain Hoechst 33342, indicating increased membrane permeability (1G37+ cells with sublethal doses of polymyxin B, which binds to lipopolysaccharide in the OM and permeabilizes the double-membrane envelope. We showed that intracellular AlamarBlue fluorescence increased as a function of polymyxin B dose (1G37-deficient cells relative to m1G37+ cells (2- to 3-fold, 1G37 deficiency was similar to the increase in E. coli cells expressing a defective OM pore protein relative to the control ( Krishnamoorthy et al., 2016 Krishnamoorthy G.

Wolloscheck D.

Weeks J.W.

Croft C.

Rybenkov V.V.

Zgurskaya H.I. Breaking the permeability barrier of Escherichia coli by controlled hyperporination of the outer membrane. Figure 3 m1G37 Deficiency Weakens the Cell Envelope Show full caption 1G37 deficiency (m1G37−) showed increased membrane permeability relative to m1G37+ cells. Cells were grown as in 1G37+ (Ara+, blue) and m1G37-deficient (Ara–, red) conditions was monitored. Levels of intracellular dye accumulation were normalized by OD 600 . Data and error bars are mean ± SD, n = 3. (A and B) E. coli (A) and Salmonella (B) trmD-KD cells in mG37 deficiency (mG37−) showed increased membrane permeability relative to mG37+ cells. Cells were grown as in Figure 2 C, and the intracellular accumulation of AlamarBlue in mG37+ (Ara+, blue) and mG37-deficient (Ara–, red) conditions was monitored. Levels of intracellular dye accumulation were normalized by OD. Data and error bars are mean ± SD, n = 3. (C–E) E. coli (C) and Salmonella (D) trmD-KD cells showed reduced Nile Red efflux in m1G37− versus m1G37+ conditions. Cells pre-loaded with Nile Red were de-energized with CCCP for 100 s, followed by addition of 50 mM glucose (Glc) to activate efflux, and the time course of Nile Red efflux was monitored for 200 s in cells grown in Ara+/− conditions. The time required to efflux 50% of the pre-loaded Nile Red (t efflux 50% ) was longer for m1G37− relative to m1G37+ cells (E). The lack of efflux in m1G37+ cells in the presence of CCCP were negative controls. Data and error bars are mean ± SD, n > 3. (F) Membrane potential was reduced in m1G37− versus m1G37+ cells as measured by ThT fluorescence. E. coli and Salmonella trmD-KD cells were inoculated in LB from a 1:100 dilution of an overnight culture without or with 0.2% Ara and grown for 4 h at 37°C, followed by dilution in LB in Ara+/− conditions to OD 600 of 0.1 and grown for 3 h at 37°C. ThT fluorescence was normalized by OD 600 . Data and error bars are mean ± SD, n > 3. (G) The population-averaged length of the cell envelope during 100-mM oscillatory osmotic shocks was shorter in Ara– (red) than Ara+ (blue) E. coli cells. Data are averaged over n > 67 cells. Inset: phase-contrast microscopy showed that E. coli trmD-KD cells were smaller in m1G37− (red) relative to m1G37+ (blue) conditions. Scale bars, 2 μm. (H) The fractional extension of the cell envelope was larger in m1G37− relative to m1G37+ cells. The extension was calculated as (l−l av )/l, where l is the effective population-averaged envelope length, and l av is the time-averaged value of l using the period of the oscillatory cycles as an averaging window. Data are averaged over n > 67 cells. (I) The amplitude of length oscillations in (H) averaged over oscillatory cycles was larger in m1G37− relative to m1G37+ cells, averaged over oscillatory cycles. Data and error bars are mean ± SD from n > 67 cells. ∗∗∗p < 0.0001 by Student’s t test. In a replicate experiment, the ratio of the amplitude of length oscillations between m1G37+ and m1G37− cells measured after sufficient m1G37 depletion to reduce growth rate to <0.2 h−1 was 1.43 (n > 609 cells). See also Figures S4 and S5 We hypothesized that the reduced biosynthesis of membrane proteins in mG37-deficient cells would damage membrane structural integrity. We observed increased intracellular accumulation in mG37-deficient bacteria of both the redox sensor AlamarBlue, which becomes fluorescent inside cells, and the DNA fluorescent stain Hoechst 33342, indicating increased membrane permeability ( Figures 3 A, 3B, and S4 A). The accumulation of each dye was measured during exponential growth, and dye exposure was initiated in the presence of carbonyl cyanide m-chlorophenyl hydrazine (CCCP) to inactivate membrane efflux. To validate that AlamarBlue fluorescence reflected the permeability of the OM, we treated E. coli and Salmonella mG37+ cells with sublethal doses of polymyxin B, which binds to lipopolysaccharide in the OM and permeabilizes the double-membrane envelope. We showed that intracellular AlamarBlue fluorescence increased as a function of polymyxin B dose ( Figure S4 B), and that the maximum increase (4- to 5-fold) at a lethal dose of polymyxin B was in the same range as the observed increases in mG37-deficient cells relative to mG37+ cells (2- to 3-fold, Figures 3 A and 3B). We further showed that the intracellular AlamarBlue increase due to mG37 deficiency was similar to the increase in E. coli cells expressing a defective OM pore protein relative to the control ( Figure S4 C). This defective pore protein was created by mutations in the siderophore transporter protein FhuA to enlarge the pore size, rendering the OM hyperpermeable to a wide range of compounds without affecting efflux ().

1G37 deficiency, we created proS-KD and cysS-KD strains, in which the essential genes responsible for amino-acid charging of tRNAPro (proS) and tRNACys (cysS), respectively, were deleted from the chromosome and cell viability was maintained by Ara-dependent, plasmid-borne expression of each native gene. The proS-KD strain was a positive control to determine whether the deficiency of Pro-tRNAPro affected translation of Pro codons in a manner similar to m1G37 deficiency, whereas the cysS-KD strain was a negative control for how depletion of an essential protein that is unlikely to be involved in OM protein biogenesis would affect membrane permeability. The relative AlamarBlue increase due to proS depletion (2- to 3-fold) was comparable to that due to m1G37 deficiency, whereas the relative change due to cysS depletion was not significant (<1.3-fold) (1G37 deficiency increases membrane permeability to the same extent as the deficiency caused by a hyperpermeable pore or by reduced levels of charged tRNA for translation of Pro codons. To further validate the significance of the AlamarBlue increase due to mG37 deficiency, we created proS-KD and cysS-KD strains, in which the essential genes responsible for amino-acid charging of tRNA(proS) and tRNA(cysS), respectively, were deleted from the chromosome and cell viability was maintained by Ara-dependent, plasmid-borne expression of each native gene. The proS-KD strain was a positive control to determine whether the deficiency of Pro-tRNAaffected translation of Pro codons in a manner similar to mG37 deficiency, whereas the cysS-KD strain was a negative control for how depletion of an essential protein that is unlikely to be involved in OM protein biogenesis would affect membrane permeability. The relative AlamarBlue increase due to proS depletion (2- to 3-fold) was comparable to that due to mG37 deficiency, whereas the relative change due to cysS depletion was not significant (<1.3-fold) ( Figure S4 C). Together, these data show that mG37 deficiency increases membrane permeability to the same extent as the deficiency caused by a hyperpermeable pore or by reduced levels of charged tRNA for translation of Pro codons.

1G37 deficiency also reduced membrane efflux, as indicated by the increased time required to pump out 50% of pre-loaded Nile Red dye (from 36 ± 1 to 66 ± 2 s for E. coli and 32 ± 3 to 45 ± 3 s for Salmonella in m1G37-deficient cells relative to m1G37+ cells, 1G37 deficiency reduces but does not eliminate levels of efflux pumps, whereas acrB deletion (ΔacrB) eliminates a component of the AcrAB-TolC complex, which is the major efflux pump responsible for expelling most antibiotics. The reduction in efflux due to m1G37 deficiency was also observed by monitoring ethidium bromide ( Prindle et al., 2015 Prindle A.

Liu J.

Asally M.

Ly S.

Garcia-Ojalvo J.

Süel G.M. Ion channels enable electrical communication in bacterial communities. 1G37 deficiency reduced the fluorescence of ThT in E. coli and Salmonella ( G37 deficiency also reduced membrane efflux, as indicated by the increased time required to pump out 50% of pre-loaded Nile Red dye (from 36 ± 1 to 66 ± 2 s for E. coli and 32 ± 3 to 45 ± 3 s for Salmonella in mG37-deficient cells relative to mG37+ cells, Figures 3 C–3E). The extensions of efflux time (1.8- and 1.4-fold for E. coli and Salmonella, respectively) were smaller than that due to the deletion of acrB relative to wild type (>4-fold) ( Figures S5 A and S5B); this smaller effect is expected because mG37 deficiency reduces but does not eliminate levels of efflux pumps, whereas acrB deletion (ΔacrB) eliminates a component of the AcrAB-TolC complex, which is the major efflux pump responsible for expelling most antibiotics. The reduction in efflux due to mG37 deficiency was also observed by monitoring ethidium bromide ( Figures S5 C and S5D), which showed an increase in the efflux time as a function of polymyxin B dose ( Figures S5 E and S5F). As expected, the extension time required for expelling ethidium bromide was smaller compared with the effect of ΔtolC on the AcrAB-TolC complex ( Figures S5 C and S5D). We also used Thioflavin T (ThT) to probe membrane potential () and confirmed that mG37 deficiency reduced the fluorescence of ThT in E. coli and Salmonella ( Figure 3 F), further supporting our conclusion that the OM was impaired.

1G37 deficiency affected the cell envelope structure, we measured cellular mechanical stiffness using an assay that we recently developed and utilized to demonstrate that the OM makes a surprisingly large contribution to the overall stiffness of the E. coli cell envelope ( Rojas et al., 2018 Rojas E.R.

Billings G.

Odermatt P.D.

Auer G.K.

Zhu L.

Miguel A.

Chang F.

Weibel D.B.

Theriot J.A.

Huang K.C. The outer membrane is an essential load-bearing element in gram-negative bacteria. Rojas et al., 2018 Rojas E.R.

Billings G.

Odermatt P.D.

Auer G.K.

Zhu L.

Miguel A.

Chang F.

Weibel D.B.

Theriot J.A.

Huang K.C. The outer membrane is an essential load-bearing element in gram-negative bacteria. Rojas et al., 2018 Rojas E.R.

Billings G.

Odermatt P.D.

Auer G.K.

Zhu L.

Miguel A.

Chang F.

Weibel D.B.

Theriot J.A.

Huang K.C. The outer membrane is an essential load-bearing element in gram-negative bacteria. Ruiz et al., 2005 Ruiz N.

Falcone B.

Kahne D.

Silhavy T.J. Chemical conditionality: a genetic strategy to probe organelle assembly. 1G37 deficiency would decrease the stiffness of the cell envelope. To determine how mG37 deficiency affected the cell envelope structure, we measured cellular mechanical stiffness using an assay that we recently developed and utilized to demonstrate that the OM makes a surprisingly large contribution to the overall stiffness of the E. coli cell envelope (). Perturbation of the OM by chemical agents or genetic mutations caused large reductions in stiffness and rendered cells susceptible to lysis under oscillatory osmotic shocks (). We previously showed that the deletion of ompA and lpp and the introduction of a mutant allele of lptD each decreased OM stiffness (). While ompA and lpp encode abundant OM proteins, the mutant lptD allele encodes a variant of the lipopolysaccharide assembly machinery that is known to increase the OM permeability to antibiotics (). We thus hypothesized that the altered OM composition during mG37 deficiency would decrease the stiffness of the cell envelope.