tRNA Methylation Is a Global Determinant of Bacterial Multi-drug Resistance

Masuda, Isao; Matsubara, Ryuma; Christian, Thomas; Liao, Jingqiu; Yadavalli, Srujana S.; Zhang, Lisheng; Goulian, Mark; Foster, Leonard J.; Huang, Kerwyn Casey; Hou, Ya‐Ming

doi:10.1016/j.cels.2019.05.002

Cited by 18 publications

(32 citation statements)

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“…These inferences were verified in several ways. The Nile Red assay for efflux 30,31 confirmed a higher rate of efflux, stimulated by AcrA (Fig. 3b).…”

Section: Dead Cells Release Acra As a Necrosignalmentioning

confidence: 59%

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Necrosignaling: Cell death triggers antibiotic survival pathways in bacterial swarms

Bhattacharyya

Walker

Harshey

2020

Preprint

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SummarySwarming is form of collective bacterial motion enabled by flagella on the surface of semi-solid media1. Many bacterial species exhibit non-genetic adaptive resistance to a broad range of antibiotics only when swarming (SR)2-4. While the swarming population as a whole survives antibiotic challenge, it nonetheless sustains considerable cell death5. In this study focused mainly on E. coli, an initial analysis of antibiotic-induced killing patterns of swarm vs planktonic cells indicated that death of a sub-population is beneficial to the swarm in promoting SR. Introduction of pre-killed cells into a swarm indeed enhanced SR, allowing us to purify the SR factor from killed cells, and to establish that cell death is directly involved in SR. The SR-enhancing factor was identified to be AcrA, a periplasmic component of a tripartite RND efflux pump6, of which the outer membrane component TolC is shared by multiple drug efflux pumps7. We show that AcrA stimulates drug efflux in live cells by interacting with TolC from the outside. This stimulus acts as a signal to activate efflux in the short term, and to induce the expression of other classes of efflux pumps in the long term, amplifying the response and establishing SR. We call this phenomenon ‘necrosignaling’, and show the existence of species-specific necrosignaling in both Gram-positive and Gram-negative bacteria. Our results have implications for the adaptive resistance of other surface-associated bacterial collectives such as biofilms. Given that such resistance is known to be an incubator for evolving genetic resistance8, our findings may also be relevant to chemotherapy-resistant cancers.

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“…These inferences were verified in several ways. The Nile Red assay for efflux 30,31 confirmed a higher rate of efflux, stimulated by AcrA (Fig. 3b).…”

Section: Dead Cells Release Acra As a Necrosignalmentioning

confidence: 59%

“…3b). The Alamar Blue assay for membrane permeability 30,32 showed lower permeability, unaffected by AcrA (Fig. 3c).…”

Section: Dead Cells Release Acra As a Necrosignalmentioning

confidence: 93%

Necrosignaling: Cell death triggers antibiotic survival pathways in bacterial swarms

Bhattacharyya

Walker

Harshey

2020

Preprint

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“…Thus, despite being placed at the same position, the chemical reactivity of acp 3 U47 to biotin-SA conjugation can differ depending on the tRNA sequence. Although both tRNA Arg/ICG and tRNA Lys/mnm5s2UUU were overexpressed in cells, the level of post-transcriptional modification to synthesize acp 3 U47 was likely stoichiometric under the expression condition, as evidenced by mass spectrometry quantification of other post-transcriptional modifications [ 40 ]. In contrast to the low reactivity of acp 3 U47, the reactivity of the alkyl-amine of the aminoacyl group after aminoacylation was robust.…”

Section: Resultsmentioning

confidence: 99%

A Label-Free Assay for Aminoacylation of tRNA

Gamper

Hou

2020

Genes

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Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical to understand the mechanism of specificity and the flux of the aa-tRNA into the protein synthesis machinery, which determines the rate of cell growth. Traditional assays for the quantification of tRNA aminoacylation involve radioactivity, either with a radioactive amino acid or with a [3′-32P]-labeled tRNA. We describe here a label-free assay that monitors aminoacylation by biotinylation-streptavidin (SA) conjugation to the α-amine or the α-imine of the aminoacyl group on the aa-tRNA. The conjugated aa-tRNA product is readily separated from the unreacted tRNA by a denaturing polyacrylamide gel, allowing for quantitative measurement of aminoacylation. This label-free assay is applicable to a wide range of amino acids and tRNA sequences and to both classes of aminoacylation. It is more sensitive and robust than the assay with a radioactive amino acid and has the potential to explore a wider range of tRNA than the assay with a [3′-32P]-labeled tRNA. This label-free assay reports kinetic parameters of aminoacylation quantitatively similar to those reported by using a radioactive amino acid, suggesting its broad applicability to research relevant to human health and disease.

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“…It is interesting that tRNA modi cation is required for cold adaptation in extreme-thermophilic bacteria [49]. In addition, it has been shown that tRNA modi cation during environmental stress is critical [50]. The gene trmD (encoding guanine-N(1)-methyltransferase) is required for multi-drug resistance in E. coli, since it provides a control mechanism to translation of membrane proteins [50].…”

Section: Translation Regulation Through Translation Initiation Factormentioning

confidence: 99%

“…In addition, it has been shown that tRNA modi cation during environmental stress is critical [50]. The gene trmD (encoding guanine-N(1)-methyltransferase) is required for multi-drug resistance in E. coli, since it provides a control mechanism to translation of membrane proteins [50]. It is possible that tRNA modi cation in the three lactic acid bacteria studied provides a similar control system for cold-shock response genes, promoting their translation.…”

Section: Translation Regulation Through Translation Initiation Factormentioning

confidence: 99%

Transcriptomic time-series analysis of cold- and heat-shock response in psychrotrophic lactic acid bacteria

Duru

Ylinen²,

Belanov³

et al. 2020

Preprint

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Background: Psychrotrophic lactic acid bacteria (LAB) species are the dominant species in microbiota of cold-stored modified-atmosphere-packaged food products and they are the main cause of food spoilage. But still, the cold- and heat-shock response of the spoilage-related psychrotrophic lactic acid bacteria has not been studied. Here, to study cold- and heat-shock response of spoilage lactic acid bacteria, we performed time-series RNA-seq for Le. gelidum, Lc. piscium and P. oligofermentans using temperatures of 0 °C, 4 °C, 14 °C, 25 °C and 28 °C. Results: We showed that the cold-shock protein A (cspA) gene was the main cold-shock protein gene among cold-shock protein genes in all three species. Our results indicated DEAD-box RNA helicase genes (cshA, cshB) play a critical role in cold-shock response in psychrotrophic LAB. In addition, several RNase genes were also involved in cold-shock response in Lc. piscium and P. oligofermentans. Moreover, gene network inference analysis provided candidate genes involved in cold-shock response. Ribosomal proteins, tRNA modification, rRNA modification, and ABC and efflux MFS transporter genes clustered with cold-shock response genes in all three species, which was a strong indication that these genes would be part of cold-shock response machinery. Heat-shock treatment caused upregulation of Clp protease and chaperone genes in all three species and we were able to identify transcription binding site motifs for heat-shock response genes in Le. gelidum and Lc. piscium. Finally, we showed that food spoilage-related genes were upregulated at cold temperatures. Conclusions: The results of this study provide new insights into a better understanding of the cold- and heat-shock response in psychrotrophic LAB. In addition, candidate genes involved in cold- and heat-shock response predicted using gene network inference analysis could be used as a target for future studies.

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tRNA Methylation Is a Global Determinant of Bacterial Multi-drug Resistance

Cited by 18 publications

References 0 publications

Necrosignaling: Cell death triggers antibiotic survival pathways in bacterial swarms

Necrosignaling: Cell death triggers antibiotic survival pathways in bacterial swarms

A Label-Free Assay for Aminoacylation of tRNA

Transcriptomic time-series analysis of cold- and heat-shock response in psychrotrophic lactic acid bacteria

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