Table 1. Open in new tab Phenotypes...

Gene	Function	Known physiological phenotypes in literature	Known effects on translation in literature	TN-seq fold change (T16 antibiotic/T16MH)—this study
tRNA modification
tgt	tRNA-guanine transglycosylase GUN anticodon tRNAs	No significant biological difference but defect in stationary phase viability (Noguchi et al. 1982)	UAG readthrough (Frey et al. 1989). Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: −15.8 (P = .002) CIP: −2 (P = .04)
gidA mnmG	5-carboxymethylamino-methyluridine-tRNA synthase. (m⁵U34)	Pleiotropic phenotypes on DNA replication, cell division, tmRNA tagging defect, reviewed in Nedialkova and Leidel (2015)	Decoding NNG codons (Kurata et al. 2008). Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: +1.9 (NS) CIP: −2.3 (P = .05)
dusA	tRNA-dihydrouridine synthase A	N/A (Bou-Nader et al. 2018)		TOB : +3.8 (P = .0002) CIP : +1.4 (NS)
dusB	tRNA-dihydrouridine synthase B	N/A (Bou-Nader et al. 2018)		TOB : +12.2 (P = 10⁻⁴) CIP : +1.3 (NS)
miaA N/A	tRNA dimethylallyltransferase (i6A37)	Mutator phenotype (Zhao et al. 2001). Reduced tetracycline resistance (Taylor et al. 1998). Evolvability of resistance to CIP (Mehi et al. 2013). Stress specific phenotype: RpoS/IraP translation (Aubee et al. 2016)	Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: −18 (P = 10⁻⁴)
miaB	Isopentenyl-adenosine A37 tRNA methylthiolase (ms2i6A37) U- starting codons	Stress specific phenotype: fur translation and iron levels (Vecerek et al. 2007)	Taylor et al. (1998)	TOB : −5.9 (P = .0007) CIP: +1.1 (NS)
truA hisT	tRNA pseudouridine38–40 synthase	Growth defect in minimal medium (Tsui et al. 1991)	Reading frame maintenance defect (Urbonavicius et al. 2001). Mistranslation (Bruni et al. 1977, Parker 1982)	TOB: +2.4 (NS) CIP: −2.1 (NS)
truB	tRNA pseudouridine55 synthase	Sensitivity to heatshock (Kinghorn et al. 2002)		TOB: −1.4 (NS) CIP: −1.9 (NS)
truC	tRNA pseudouridine65 synthase tRNAIle1 and tRNAAsp	N/A (Del Campo et al. 2001)		TOB: 1 CIP: 1
trmA	tRNA m⁵U54 methyltransferase and Y341 of tmRNA	N/A (Bjork and Neidhardt 1975). Sensitivity to heatshock of truB trmA (Kinghorn et al. 2002).		TOB: −2.3 (P = .003) CIP: −1.2 (NS)
trmB	tRNA m⁷G46 methyltransferase	N/A (De Bie et al. 2003)		TOB: +2.7 (P = .008) CIP: +3.4 (p = 0.06 NS)
trmEmnmE	5-carboxymethylaminomethyluridine-tRNA synthase m⁵U modification of U34 in tRNA	Pleiotropic, acts with gidA, see gidA above	Reading frame maintenance defect (Urbonavicius et al. 2001). UAG readthrough (Elseviers et al. 1984). Mistranslation (Hagervall et al. 1998)	TOB: +3.9 (P = .01) CIP: +2.4 (NS)
trmH	tRNA (Gm18) 2'-O-methyltransferase	N/A (Persson et al. 1997)		TOB : +5.4 (P = .0002) CIP : −1.3 (NS)
trmK	tRNA (m¹A22)methyltransferase	Kimura et al. (2020)		TOB : 1 CIP : −1.3 (NS)
*rRNA modification (positions described in Escherichia coli)*
rlmB	23S rRNA 2'-O-ribose G2251 methyltransferase	No obvious growth defect (Lovgren and Wikstrom 2001)		TOB: −2.6 (P = .001) CIP: +2 (P = .05)
rlmI	23S rRNA m5C1962 methyltransferase	Decrease in biofilm formation (Herzberg et al. 2006). Slight growth defect (Purta et al. 2008b).		TOB: −1.4 (NS) CIP: +1.3 (NS)
rlmH N/A	23S rRNA m3Ψ1915 methyltransferase	Slight growth defect (Purta et al. 2008a)		TOB: −3 (P = .003) CIP: −1.3 (NS)
rlmE/rrmJ N/A	23S rRNA 2'-O-ribose U2552 methyltransferase	Decreased growth rate (Caldas et al. 2000, Toh and Mankin 2008, Pletnev et al. 2020). Sparsomycin and tiamulin sensitive (Toh and Mankin 2008). Lincomycin sentitive (Caldas et al. 2000).	Frameshift and stop codon readthrough (Widerak et al. 2005). Accumulation of ribosomal subunit intermediates (Pletnev et al. 2020)	TOB: +1.2 (NS) CIP: +825 (P = .003)
rsmB	16S rRNA m⁵C967 methyltransferase	No obvious growth defect (Gu et al. 1999, Pletnev et al. 2020)	Accumulation of 17S rRNA (Pletnev et al. 2020). Translation initiation (Burakovsky et al. 2012, Arora et al. 2013)	TOB: −6.7 (P = .007) CIP: −2.9 NS
rsmC	16S rRNA m²G1207 methyltransferase	No obvious growth defect (Pletnev et al. 2020)	Correct folding of 16S rRNA (Gc et al. 2020)	TOB: +2.3 (P = .05) CIP: −1.2 (NS)
rsmD	16S rRNA m²G966 methyltransferase	No obvious growth defect (Lesnyak et al. 2007, Pletnev et al. 2020)	Translation initiation (Burakovsky et al. 2012, Arora et al. 2013).	TOB: −5.7 (P = .006) CIP: 1.1 (NS)
rsmF/yebU	16S rRNA m⁵C1407 methyltransferase	No obvious (Pletnev et al. 2020) or slight (Andersen and Douthwaite 2006) growth defect Increased resistance to some AGs reported (Gutierrez et al. 2012)	Role in translation initiation (Das et al. 2008)	TOB: −2.5 (P = .002) CIP: −1.8 (NS)
rsmG N/A	16S rRNA m7G527 methyltransferase	Mutations found in streptomycin resistant MTB clinical isolates (Okamoto et al. 2007)		TOB: −7.7 (P = .0009)
rsmH N/A	16S rRNA m⁴C1402 methyltransferase	No obvious growth defect (Dassain et al. 1999) ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)	Decoding fidelity (Kimura and Suzuki 2010)	TOB: −8.4 (P = .0009)
rsmI N/A	16S rRNA 2'-O-ribose C1402 methyltransferase	No obvious growth defect ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)	Decoding fidelity (Kimura and Suzuki 2010)	TOB: −8.4 (P = 10⁻⁴)
rsuA	16S rRNA pseudouridine516 synthase	No obvious growth defect (Conrad et al. 1999) Overexpression leads to resistance to HOCl (Chen et al. 2021)	Accumulation of 17S rRNA (the present study)	TOB: −2.3 (P = .01) CIP: 1
rluB	23S rRNA pseudouridine2605 synthase	No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008). Increased CM and linezolid sensitivity (Toh and Mankin 2008)	50S subunit maturation (Jiang et al. 2007)	TOB: +9.4 (P = 10⁻⁴) CIP: +1.2 (NS)
rluC N/A	23S rRNA pseudouridine955/2504/2580 synthase	No obvious growth defect (Conrad et al. 1998). Cold sensitivity (Jiang et al. 2007) clindamycin, linezolid, and tiamulin sensitivity (Toh and Mankin 2008)		TOB: +1.8 (P = 10⁻⁴) CIP: +1.3 (NS)
rluD	23S rRNA pseudouridine1911/1915/1917 synthase	Reported to cause a large growth defect in Escherichia coli but independently of pseudourdines (Gutgsell et al. 2001)	Ribosome assembly (Gutgsell et al. 2005)	TOB: +1.4 (P = .03) CIP: +6.6 (P = .002)
rluE	23S rRNA pseudouridine2457 synthase	No obvious growth defect (Del Campo et al. 2001)		TOB: −1.5 (P = .002) CIP: −2.4 (NS)
Modification of both tRNA and rRNA
rluF	23S rRNA pseudouridine2604/tRNATyr pseudouridine35 synthase	No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008, Pletnev et al. 2020). Decreased linezolid resistance (Toh and Mankin 2008)	Effect on translation of Tyr codons (Addepalli and Limbach 2016)	TOB: −1.2 (NS) CIP: −1.2 (NS)
rlmN	tRNA m2A37 methyltransferase/23S rRNA m2A2503 methyltransferase	No obvious growth defect (Benitez-Paez et al. 2012). Slightly increased susceptibility to certain peptidyl transferase-targeting antibiotics (Toh and Mankin 2008)	UAG readthrough (Benitez-Paez et al. 2012)	TOB: +2.3 (P = .005) CIP: −2.2 (P = .0003)

Gene

Function

Known physiological phenotypes in literature

Known effects on translation in literature

TN-seq fold change (T16 antibiotic/T16MH)—this study

tRNA modification

tgt

tRNA-guanine transglycosylase GUN anticodon tRNAs

No significant biological difference but defect in stationary phase viability (Noguchi et al. 1982)

UAG readthrough (Frey et al. 1989). Reading frame maintenance defect (Urbonavicius et al. 2001)

TOB: −15.8 (P = .002) CIP: −2 (P = .04)

gidA mnmG

5-carboxymethylamino-methyluridine-tRNA synthase. (m⁵U34)

Pleiotropic phenotypes on DNA replication, cell division, tmRNA tagging defect, reviewed in Nedialkova and Leidel (2015)

Decoding NNG codons (Kurata et al. 2008). Reading frame maintenance defect (Urbonavicius et al. 2001)

TOB: +1.9 (NS) CIP: −2.3 (P = .05)

dusA

tRNA-dihydrouridine synthase A

N/A (Bou-Nader et al. 2018)

TOB : +3.8 (P = .0002) CIP : +1.4 (NS)

dusB

tRNA-dihydrouridine synthase B

N/A (Bou-Nader et al. 2018)

TOB : +12.2 (P = 10⁻⁴) CIP : +1.3 (NS)

miaA N/A

tRNA dimethylallyltransferase (i6A37)

Mutator phenotype (Zhao et al. 2001). Reduced tetracycline resistance (Taylor et al. 1998). Evolvability of resistance to CIP (Mehi et al. 2013). Stress specific phenotype: RpoS/IraP translation (Aubee et al. 2016)

Reading frame maintenance defect (Urbonavicius et al. 2001)

TOB: −18 (P = 10⁻⁴)

miaB

Isopentenyl-adenosine A37 tRNA methylthiolase (ms2i6A37) U- starting codons

Stress specific phenotype: fur translation and iron levels (Vecerek et al. 2007)

Taylor et al. (1998)

TOB : −5.9 (P = .0007) CIP: +1.1 (NS)

truA hisT

tRNA pseudouridine38–40 synthase

Growth defect in minimal medium (Tsui et al. 1991)

Reading frame maintenance defect (Urbonavicius et al. 2001). Mistranslation (Bruni et al. 1977, Parker 1982)

TOB: +2.4 (NS) CIP: −2.1 (NS)

truB

tRNA pseudouridine55 synthase

Sensitivity to heatshock (Kinghorn et al. 2002)

TOB: −1.4 (NS) CIP: −1.9 (NS)

truC

tRNA pseudouridine65 synthase tRNAIle1 and tRNAAsp

N/A (Del Campo et al. 2001)

TOB: 1 CIP: 1

trmA

tRNA m⁵U54 methyltransferase and Y341 of tmRNA

N/A (Bjork and Neidhardt 1975). Sensitivity to heatshock of truB trmA (Kinghorn et al. 2002).

TOB: −2.3 (P = .003) CIP: −1.2 (NS)

trmB

tRNA m⁷G46 methyltransferase

N/A (De Bie et al. 2003)

TOB: +2.7 (P = .008) CIP: +3.4 (p = 0.06 NS)

trmEmnmE

5-carboxymethylaminomethyluridine-tRNA synthase m⁵U modification of U34 in tRNA

Pleiotropic, acts with gidA, see gidA above

Reading frame maintenance defect (Urbonavicius et al. 2001). UAG readthrough (Elseviers et al. 1984). Mistranslation (Hagervall et al. 1998)

TOB: +3.9 (P = .01) CIP: +2.4 (NS)

trmH

tRNA (Gm18) 2'-O-methyltransferase

N/A (Persson et al. 1997)

TOB : +5.4 (P = .0002) CIP : −1.3 (NS)

trmK

tRNA (m¹A22)methyltransferase

Kimura et al. (2020)

TOB : 1 CIP : −1.3 (NS)

rRNA modification (positions described in Escherichia coli)

rlmB

23S rRNA 2'-O-ribose G2251 methyltransferase

No obvious growth defect (Lovgren and Wikstrom 2001)

TOB: −2.6 (P = .001) CIP: +2 (P = .05)

rlmI

23S rRNA m5C1962 methyltransferase

Decrease in biofilm formation (Herzberg et al. 2006). Slight growth defect (Purta et al. 2008b).

TOB: −1.4 (NS) CIP: +1.3 (NS)

rlmH N/A

23S rRNA m3Ψ1915 methyltransferase

Slight growth defect (Purta et al. 2008a)

TOB: −3 (P = .003) CIP: −1.3 (NS)

rlmE/rrmJ N/A

23S rRNA 2'-O-ribose U2552 methyltransferase

Decreased growth rate (Caldas et al. 2000, Toh and Mankin 2008, Pletnev et al. 2020). Sparsomycin and tiamulin sensitive (Toh and Mankin 2008). Lincomycin sentitive (Caldas et al. 2000).

Frameshift and stop codon readthrough (Widerak et al. 2005). Accumulation of ribosomal subunit intermediates (Pletnev et al. 2020)

TOB: +1.2 (NS) CIP: +825 (P = .003)

rsmB

16S rRNA m⁵C967 methyltransferase

No obvious growth defect (Gu et al. 1999, Pletnev et al. 2020)

Accumulation of 17S rRNA (Pletnev et al. 2020). Translation initiation (Burakovsky et al. 2012, Arora et al. 2013)

TOB: −6.7 (P = .007) CIP: −2.9 NS

rsmC

16S rRNA m²G1207 methyltransferase

No obvious growth defect (Pletnev et al. 2020)

Correct folding of 16S rRNA (Gc et al. 2020)

TOB: +2.3 (P = .05) CIP: −1.2 (NS)

rsmD

16S rRNA m²G966 methyltransferase

No obvious growth defect (Lesnyak et al. 2007, Pletnev et al. 2020)

Translation initiation (Burakovsky et al. 2012, Arora et al. 2013).

TOB: −5.7 (P = .006) CIP: 1.1 (NS)

rsmF/yebU

16S rRNA m⁵C1407 methyltransferase

No obvious (Pletnev et al. 2020) or slight (Andersen and Douthwaite 2006) growth defect Increased resistance to some AGs reported (Gutierrez et al. 2012)

Role in translation initiation (Das et al. 2008)

TOB: −2.5 (P = .002) CIP: −1.8 (NS)

rsmG N/A

16S rRNA m7G527 methyltransferase

Mutations found in streptomycin resistant MTB clinical isolates (Okamoto et al. 2007)

TOB: −7.7 (P = .0009)

rsmH N/A

16S rRNA m⁴C1402 methyltransferase

No obvious growth defect (Dassain et al. 1999) ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)

Decoding fidelity (Kimura and Suzuki 2010)

TOB: −8.4 (P = .0009)

rsmI N/A

16S rRNA 2'-O-ribose C1402 methyltransferase

No obvious growth defect ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)

Decoding fidelity (Kimura and Suzuki 2010)

TOB: −8.4 (P = 10⁻⁴)

rsuA

16S rRNA pseudouridine516 synthase

No obvious growth defect (Conrad et al. 1999) Overexpression leads to resistance to HOCl (Chen et al. 2021)

Accumulation of 17S rRNA (the present study)

TOB: −2.3 (P = .01) CIP: 1

rluB

23S rRNA pseudouridine2605 synthase

No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008). Increased CM and linezolid sensitivity (Toh and Mankin 2008)

50S subunit maturation (Jiang et al. 2007)

TOB: +9.4 (P = 10⁻⁴) CIP: +1.2 (NS)

rluC N/A

23S rRNA pseudouridine955/2504/2580 synthase

No obvious growth defect (Conrad et al. 1998). Cold sensitivity (Jiang et al. 2007) clindamycin, linezolid, and tiamulin sensitivity (Toh and Mankin 2008)

TOB: +1.8 (P = 10⁻⁴) CIP: +1.3 (NS)

rluD

23S rRNA pseudouridine1911/1915/1917 synthase

Reported to cause a large growth defect in Escherichia coli but independently of pseudourdines (Gutgsell et al. 2001)

Ribosome assembly (Gutgsell et al. 2005)

TOB: +1.4 (P = .03) CIP: +6.6 (P = .002)

rluE

23S rRNA pseudouridine2457 synthase

No obvious growth defect (Del Campo et al. 2001)

TOB: −1.5 (P = .002) CIP: −2.4 (NS)

Modification of both tRNA and rRNA

rluF

23S rRNA pseudouridine2604/tRNATyr pseudouridine35 synthase

No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008, Pletnev et al. 2020). Decreased linezolid resistance (Toh and Mankin 2008)

Effect on translation of Tyr codons (Addepalli and Limbach 2016)

TOB: −1.2 (NS) CIP: −1.2 (NS)

rlmN

tRNA m2A37 methyltransferase/23S rRNA m2A2503 methyltransferase

No obvious growth defect (Benitez-Paez et al. 2012). Slightly increased susceptibility to certain peptidyl transferase-targeting antibiotics (Toh and Mankin 2008)

UAG readthrough (Benitez-Paez et al. 2012)

TOB: +2.3 (P = .005) CIP: −2.2 (P = .0003)

Gene	Function	Known physiological phenotypes in literature	Known effects on translation in literature	TN-seq fold change (T16 antibiotic/T16MH)—this study
tRNA modification
tgt	tRNA-guanine transglycosylase GUN anticodon tRNAs	No significant biological difference but defect in stationary phase viability (Noguchi et al. 1982)	UAG readthrough (Frey et al. 1989). Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: −15.8 (P = .002) CIP: −2 (P = .04)
gidA mnmG	5-carboxymethylamino-methyluridine-tRNA synthase. (m⁵U34)	Pleiotropic phenotypes on DNA replication, cell division, tmRNA tagging defect, reviewed in Nedialkova and Leidel (2015)	Decoding NNG codons (Kurata et al. 2008). Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: +1.9 (NS) CIP: −2.3 (P = .05)
dusA	tRNA-dihydrouridine synthase A	N/A (Bou-Nader et al. 2018)		TOB : +3.8 (P = .0002) CIP : +1.4 (NS)
dusB	tRNA-dihydrouridine synthase B	N/A (Bou-Nader et al. 2018)		TOB : +12.2 (P = 10⁻⁴) CIP : +1.3 (NS)
miaA N/A	tRNA dimethylallyltransferase (i6A37)	Mutator phenotype (Zhao et al. 2001). Reduced tetracycline resistance (Taylor et al. 1998). Evolvability of resistance to CIP (Mehi et al. 2013). Stress specific phenotype: RpoS/IraP translation (Aubee et al. 2016)	Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: −18 (P = 10⁻⁴)
miaB	Isopentenyl-adenosine A37 tRNA methylthiolase (ms2i6A37) U- starting codons	Stress specific phenotype: fur translation and iron levels (Vecerek et al. 2007)	Taylor et al. (1998)	TOB : −5.9 (P = .0007) CIP: +1.1 (NS)
truA hisT	tRNA pseudouridine38–40 synthase	Growth defect in minimal medium (Tsui et al. 1991)	Reading frame maintenance defect (Urbonavicius et al. 2001). Mistranslation (Bruni et al. 1977, Parker 1982)	TOB: +2.4 (NS) CIP: −2.1 (NS)
truB	tRNA pseudouridine55 synthase	Sensitivity to heatshock (Kinghorn et al. 2002)		TOB: −1.4 (NS) CIP: −1.9 (NS)
truC	tRNA pseudouridine65 synthase tRNAIle1 and tRNAAsp	N/A (Del Campo et al. 2001)		TOB: 1 CIP: 1
trmA	tRNA m⁵U54 methyltransferase and Y341 of tmRNA	N/A (Bjork and Neidhardt 1975). Sensitivity to heatshock of truB trmA (Kinghorn et al. 2002).		TOB: −2.3 (P = .003) CIP: −1.2 (NS)
trmB	tRNA m⁷G46 methyltransferase	N/A (De Bie et al. 2003)		TOB: +2.7 (P = .008) CIP: +3.4 (p = 0.06 NS)
trmEmnmE	5-carboxymethylaminomethyluridine-tRNA synthase m⁵U modification of U34 in tRNA	Pleiotropic, acts with gidA, see gidA above	Reading frame maintenance defect (Urbonavicius et al. 2001). UAG readthrough (Elseviers et al. 1984). Mistranslation (Hagervall et al. 1998)	TOB: +3.9 (P = .01) CIP: +2.4 (NS)
trmH	tRNA (Gm18) 2'-O-methyltransferase	N/A (Persson et al. 1997)		TOB : +5.4 (P = .0002) CIP : −1.3 (NS)
trmK	tRNA (m¹A22)methyltransferase	Kimura et al. (2020)		TOB : 1 CIP : −1.3 (NS)
*rRNA modification (positions described in Escherichia coli)*
rlmB	23S rRNA 2'-O-ribose G2251 methyltransferase	No obvious growth defect (Lovgren and Wikstrom 2001)		TOB: −2.6 (P = .001) CIP: +2 (P = .05)
rlmI	23S rRNA m5C1962 methyltransferase	Decrease in biofilm formation (Herzberg et al. 2006). Slight growth defect (Purta et al. 2008b).		TOB: −1.4 (NS) CIP: +1.3 (NS)
rlmH N/A	23S rRNA m3Ψ1915 methyltransferase	Slight growth defect (Purta et al. 2008a)		TOB: −3 (P = .003) CIP: −1.3 (NS)
rlmE/rrmJ N/A	23S rRNA 2'-O-ribose U2552 methyltransferase	Decreased growth rate (Caldas et al. 2000, Toh and Mankin 2008, Pletnev et al. 2020). Sparsomycin and tiamulin sensitive (Toh and Mankin 2008). Lincomycin sentitive (Caldas et al. 2000).	Frameshift and stop codon readthrough (Widerak et al. 2005). Accumulation of ribosomal subunit intermediates (Pletnev et al. 2020)	TOB: +1.2 (NS) CIP: +825 (P = .003)
rsmB	16S rRNA m⁵C967 methyltransferase	No obvious growth defect (Gu et al. 1999, Pletnev et al. 2020)	Accumulation of 17S rRNA (Pletnev et al. 2020). Translation initiation (Burakovsky et al. 2012, Arora et al. 2013)	TOB: −6.7 (P = .007) CIP: −2.9 NS
rsmC	16S rRNA m²G1207 methyltransferase	No obvious growth defect (Pletnev et al. 2020)	Correct folding of 16S rRNA (Gc et al. 2020)	TOB: +2.3 (P = .05) CIP: −1.2 (NS)
rsmD	16S rRNA m²G966 methyltransferase	No obvious growth defect (Lesnyak et al. 2007, Pletnev et al. 2020)	Translation initiation (Burakovsky et al. 2012, Arora et al. 2013).	TOB: −5.7 (P = .006) CIP: 1.1 (NS)
rsmF/yebU	16S rRNA m⁵C1407 methyltransferase	No obvious (Pletnev et al. 2020) or slight (Andersen and Douthwaite 2006) growth defect Increased resistance to some AGs reported (Gutierrez et al. 2012)	Role in translation initiation (Das et al. 2008)	TOB: −2.5 (P = .002) CIP: −1.8 (NS)
rsmG N/A	16S rRNA m7G527 methyltransferase	Mutations found in streptomycin resistant MTB clinical isolates (Okamoto et al. 2007)		TOB: −7.7 (P = .0009)
rsmH N/A	16S rRNA m⁴C1402 methyltransferase	No obvious growth defect (Dassain et al. 1999) ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)	Decoding fidelity (Kimura and Suzuki 2010)	TOB: −8.4 (P = .0009)
rsmI N/A	16S rRNA 2'-O-ribose C1402 methyltransferase	No obvious growth defect ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)	Decoding fidelity (Kimura and Suzuki 2010)	TOB: −8.4 (P = 10⁻⁴)
rsuA	16S rRNA pseudouridine516 synthase	No obvious growth defect (Conrad et al. 1999) Overexpression leads to resistance to HOCl (Chen et al. 2021)	Accumulation of 17S rRNA (the present study)	TOB: −2.3 (P = .01) CIP: 1
rluB	23S rRNA pseudouridine2605 synthase	No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008). Increased CM and linezolid sensitivity (Toh and Mankin 2008)	50S subunit maturation (Jiang et al. 2007)	TOB: +9.4 (P = 10⁻⁴) CIP: +1.2 (NS)
rluC N/A	23S rRNA pseudouridine955/2504/2580 synthase	No obvious growth defect (Conrad et al. 1998). Cold sensitivity (Jiang et al. 2007) clindamycin, linezolid, and tiamulin sensitivity (Toh and Mankin 2008)		TOB: +1.8 (P = 10⁻⁴) CIP: +1.3 (NS)
rluD	23S rRNA pseudouridine1911/1915/1917 synthase	Reported to cause a large growth defect in Escherichia coli but independently of pseudourdines (Gutgsell et al. 2001)	Ribosome assembly (Gutgsell et al. 2005)	TOB: +1.4 (P = .03) CIP: +6.6 (P = .002)
rluE	23S rRNA pseudouridine2457 synthase	No obvious growth defect (Del Campo et al. 2001)		TOB: −1.5 (P = .002) CIP: −2.4 (NS)
Modification of both tRNA and rRNA
rluF	23S rRNA pseudouridine2604/tRNATyr pseudouridine35 synthase	No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008, Pletnev et al. 2020). Decreased linezolid resistance (Toh and Mankin 2008)	Effect on translation of Tyr codons (Addepalli and Limbach 2016)	TOB: −1.2 (NS) CIP: −1.2 (NS)
rlmN	tRNA m2A37 methyltransferase/23S rRNA m2A2503 methyltransferase	No obvious growth defect (Benitez-Paez et al. 2012). Slightly increased susceptibility to certain peptidyl transferase-targeting antibiotics (Toh and Mankin 2008)	UAG readthrough (Benitez-Paez et al. 2012)	TOB: +2.3 (P = .005) CIP: −2.2 (P = .0003)

Gene

Function

Known physiological phenotypes in literature

Known effects on translation in literature

TN-seq fold change (T16 antibiotic/T16MH)—this study

tRNA modification

tgt

tRNA-guanine transglycosylase GUN anticodon tRNAs

No significant biological difference but defect in stationary phase viability (Noguchi et al. 1982)

UAG readthrough (Frey et al. 1989). Reading frame maintenance defect (Urbonavicius et al. 2001)

TOB: −15.8 (P = .002) CIP: −2 (P = .04)

gidA mnmG

5-carboxymethylamino-methyluridine-tRNA synthase. (m⁵U34)

Pleiotropic phenotypes on DNA replication, cell division, tmRNA tagging defect, reviewed in Nedialkova and Leidel (2015)

Decoding NNG codons (Kurata et al. 2008). Reading frame maintenance defect (Urbonavicius et al. 2001)

TOB: +1.9 (NS) CIP: −2.3 (P = .05)

dusA

tRNA-dihydrouridine synthase A

N/A (Bou-Nader et al. 2018)

TOB : +3.8 (P = .0002) CIP : +1.4 (NS)

dusB

tRNA-dihydrouridine synthase B

N/A (Bou-Nader et al. 2018)

TOB : +12.2 (P = 10⁻⁴) CIP : +1.3 (NS)

miaA N/A

tRNA dimethylallyltransferase (i6A37)

Reading frame maintenance defect (Urbonavicius et al. 2001)

TOB: −18 (P = 10⁻⁴)

miaB

Isopentenyl-adenosine A37 tRNA methylthiolase (ms2i6A37) U- starting codons

Stress specific phenotype: fur translation and iron levels (Vecerek et al. 2007)

Taylor et al. (1998)

TOB : −5.9 (P = .0007) CIP: +1.1 (NS)

truA hisT

tRNA pseudouridine38–40 synthase

Growth defect in minimal medium (Tsui et al. 1991)

Reading frame maintenance defect (Urbonavicius et al. 2001). Mistranslation (Bruni et al. 1977, Parker 1982)

TOB: +2.4 (NS) CIP: −2.1 (NS)

truB

tRNA pseudouridine55 synthase

Sensitivity to heatshock (Kinghorn et al. 2002)

TOB: −1.4 (NS) CIP: −1.9 (NS)

truC

tRNA pseudouridine65 synthase tRNAIle1 and tRNAAsp

N/A (Del Campo et al. 2001)

TOB: 1 CIP: 1

trmA

tRNA m⁵U54 methyltransferase and Y341 of tmRNA

N/A (Bjork and Neidhardt 1975). Sensitivity to heatshock of truB trmA (Kinghorn et al. 2002).

TOB: −2.3 (P = .003) CIP: −1.2 (NS)

trmB

tRNA m⁷G46 methyltransferase

N/A (De Bie et al. 2003)

TOB: +2.7 (P = .008) CIP: +3.4 (p = 0.06 NS)

trmEmnmE

5-carboxymethylaminomethyluridine-tRNA synthase m⁵U modification of U34 in tRNA

Pleiotropic, acts with gidA, see gidA above

Reading frame maintenance defect (Urbonavicius et al. 2001). UAG readthrough (Elseviers et al. 1984). Mistranslation (Hagervall et al. 1998)

TOB: +3.9 (P = .01) CIP: +2.4 (NS)

trmH

tRNA (Gm18) 2'-O-methyltransferase

N/A (Persson et al. 1997)

TOB : +5.4 (P = .0002) CIP : −1.3 (NS)

trmK

tRNA (m¹A22)methyltransferase

Kimura et al. (2020)

TOB : 1 CIP : −1.3 (NS)

rRNA modification (positions described in Escherichia coli)

rlmB

23S rRNA 2'-O-ribose G2251 methyltransferase

No obvious growth defect (Lovgren and Wikstrom 2001)

TOB: −2.6 (P = .001) CIP: +2 (P = .05)

rlmI

23S rRNA m5C1962 methyltransferase

Decrease in biofilm formation (Herzberg et al. 2006). Slight growth defect (Purta et al. 2008b).

TOB: −1.4 (NS) CIP: +1.3 (NS)

rlmH N/A

23S rRNA m3Ψ1915 methyltransferase

Slight growth defect (Purta et al. 2008a)

TOB: −3 (P = .003) CIP: −1.3 (NS)

rlmE/rrmJ N/A

23S rRNA 2'-O-ribose U2552 methyltransferase

Decreased growth rate (Caldas et al. 2000, Toh and Mankin 2008, Pletnev et al. 2020). Sparsomycin and tiamulin sensitive (Toh and Mankin 2008). Lincomycin sentitive (Caldas et al. 2000).

Frameshift and stop codon readthrough (Widerak et al. 2005). Accumulation of ribosomal subunit intermediates (Pletnev et al. 2020)

TOB: +1.2 (NS) CIP: +825 (P = .003)

rsmB

16S rRNA m⁵C967 methyltransferase

No obvious growth defect (Gu et al. 1999, Pletnev et al. 2020)

Accumulation of 17S rRNA (Pletnev et al. 2020). Translation initiation (Burakovsky et al. 2012, Arora et al. 2013)

TOB: −6.7 (P = .007) CIP: −2.9 NS

rsmC

16S rRNA m²G1207 methyltransferase

No obvious growth defect (Pletnev et al. 2020)

Correct folding of 16S rRNA (Gc et al. 2020)

TOB: +2.3 (P = .05) CIP: −1.2 (NS)

rsmD

16S rRNA m²G966 methyltransferase

No obvious growth defect (Lesnyak et al. 2007, Pletnev et al. 2020)

Translation initiation (Burakovsky et al. 2012, Arora et al. 2013).

TOB: −5.7 (P = .006) CIP: 1.1 (NS)

rsmF/yebU

16S rRNA m⁵C1407 methyltransferase

No obvious (Pletnev et al. 2020) or slight (Andersen and Douthwaite 2006) growth defect Increased resistance to some AGs reported (Gutierrez et al. 2012)

Role in translation initiation (Das et al. 2008)

TOB: −2.5 (P = .002) CIP: −1.8 (NS)

rsmG N/A

16S rRNA m7G527 methyltransferase

Mutations found in streptomycin resistant MTB clinical isolates (Okamoto et al. 2007)

TOB: −7.7 (P = .0009)

rsmH N/A

16S rRNA m⁴C1402 methyltransferase

No obvious growth defect (Dassain et al. 1999) ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)

Decoding fidelity (Kimura and Suzuki 2010)

TOB: −8.4 (P = .0009)

rsmI N/A

16S rRNA 2'-O-ribose C1402 methyltransferase

No obvious growth defect ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)

Decoding fidelity (Kimura and Suzuki 2010)

TOB: −8.4 (P = 10⁻⁴)

rsuA

16S rRNA pseudouridine516 synthase

No obvious growth defect (Conrad et al. 1999) Overexpression leads to resistance to HOCl (Chen et al. 2021)

Accumulation of 17S rRNA (the present study)

TOB: −2.3 (P = .01) CIP: 1

rluB

23S rRNA pseudouridine2605 synthase

No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008). Increased CM and linezolid sensitivity (Toh and Mankin 2008)

50S subunit maturation (Jiang et al. 2007)

TOB: +9.4 (P = 10⁻⁴) CIP: +1.2 (NS)

rluC N/A

23S rRNA pseudouridine955/2504/2580 synthase

No obvious growth defect (Conrad et al. 1998). Cold sensitivity (Jiang et al. 2007) clindamycin, linezolid, and tiamulin sensitivity (Toh and Mankin 2008)

TOB: +1.8 (P = 10⁻⁴) CIP: +1.3 (NS)

rluD

23S rRNA pseudouridine1911/1915/1917 synthase

Reported to cause a large growth defect in Escherichia coli but independently of pseudourdines (Gutgsell et al. 2001)

Ribosome assembly (Gutgsell et al. 2005)

TOB: +1.4 (P = .03) CIP: +6.6 (P = .002)

rluE

23S rRNA pseudouridine2457 synthase

No obvious growth defect (Del Campo et al. 2001)

TOB: −1.5 (P = .002) CIP: −2.4 (NS)

Modification of both tRNA and rRNA

rluF

23S rRNA pseudouridine2604/tRNATyr pseudouridine35 synthase

No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008, Pletnev et al. 2020). Decreased linezolid resistance (Toh and Mankin 2008)

Effect on translation of Tyr codons (Addepalli and Limbach 2016)

TOB: −1.2 (NS) CIP: −1.2 (NS)

rlmN

tRNA m2A37 methyltransferase/23S rRNA m2A2503 methyltransferase

No obvious growth defect (Benitez-Paez et al. 2012). Slightly increased susceptibility to certain peptidyl transferase-targeting antibiotics (Toh and Mankin 2008)

UAG readthrough (Benitez-Paez et al. 2012)

TOB: +2.3 (P = .005) CIP: −2.2 (P = .0003)

Table 1.

Open in new tab

Phenotypes associated to RNA modification genes identified by TN-seq. “N/A” means: not selected for further study. NS: nonsignificant P-value.

Gene	Function	Known physiological phenotypes in literature	Known effects on translation in literature	TN-seq fold change (T16 antibiotic/T16MH)—this study
tRNA modification
tgt	tRNA-guanine transglycosylase GUN anticodon tRNAs	No significant biological difference but defect in stationary phase viability (Noguchi et al. 1982)	UAG readthrough (Frey et al. 1989). Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: −15.8 (P = .002) CIP: −2 (P = .04)
gidA mnmG	5-carboxymethylamino-methyluridine-tRNA synthase. (m⁵U34)	Pleiotropic phenotypes on DNA replication, cell division, tmRNA tagging defect, reviewed in Nedialkova and Leidel (2015)	Decoding NNG codons (Kurata et al. 2008). Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: +1.9 (NS) CIP: −2.3 (P = .05)
dusA	tRNA-dihydrouridine synthase A	N/A (Bou-Nader et al. 2018)		TOB : +3.8 (P = .0002) CIP : +1.4 (NS)
dusB	tRNA-dihydrouridine synthase B	N/A (Bou-Nader et al. 2018)		TOB : +12.2 (P = 10⁻⁴) CIP : +1.3 (NS)
miaA N/A	tRNA dimethylallyltransferase (i6A37)	Mutator phenotype (Zhao et al. 2001). Reduced tetracycline resistance (Taylor et al. 1998). Evolvability of resistance to CIP (Mehi et al. 2013). Stress specific phenotype: RpoS/IraP translation (Aubee et al. 2016)	Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: −18 (P = 10⁻⁴)
miaB	Isopentenyl-adenosine A37 tRNA methylthiolase (ms2i6A37) U- starting codons	Stress specific phenotype: fur translation and iron levels (Vecerek et al. 2007)	Taylor et al. (1998)	TOB : −5.9 (P = .0007) CIP: +1.1 (NS)
truA hisT	tRNA pseudouridine38–40 synthase	Growth defect in minimal medium (Tsui et al. 1991)	Reading frame maintenance defect (Urbonavicius et al. 2001). Mistranslation (Bruni et al. 1977, Parker 1982)	TOB: +2.4 (NS) CIP: −2.1 (NS)
truB	tRNA pseudouridine55 synthase	Sensitivity to heatshock (Kinghorn et al. 2002)		TOB: −1.4 (NS) CIP: −1.9 (NS)
truC	tRNA pseudouridine65 synthase tRNAIle1 and tRNAAsp	N/A (Del Campo et al. 2001)		TOB: 1 CIP: 1
trmA	tRNA m⁵U54 methyltransferase and Y341 of tmRNA	N/A (Bjork and Neidhardt 1975). Sensitivity to heatshock of truB trmA (Kinghorn et al. 2002).		TOB: −2.3 (P = .003) CIP: −1.2 (NS)
trmB	tRNA m⁷G46 methyltransferase	N/A (De Bie et al. 2003)		TOB: +2.7 (P = .008) CIP: +3.4 (p = 0.06 NS)
trmEmnmE	5-carboxymethylaminomethyluridine-tRNA synthase m⁵U modification of U34 in tRNA	Pleiotropic, acts with gidA, see gidA above	Reading frame maintenance defect (Urbonavicius et al. 2001). UAG readthrough (Elseviers et al. 1984). Mistranslation (Hagervall et al. 1998)	TOB: +3.9 (P = .01) CIP: +2.4 (NS)
trmH	tRNA (Gm18) 2'-O-methyltransferase	N/A (Persson et al. 1997)		TOB : +5.4 (P = .0002) CIP : −1.3 (NS)
trmK	tRNA (m¹A22)methyltransferase	Kimura et al. (2020)		TOB : 1 CIP : −1.3 (NS)
*rRNA modification (positions described in Escherichia coli)*
rlmB	23S rRNA 2'-O-ribose G2251 methyltransferase	No obvious growth defect (Lovgren and Wikstrom 2001)		TOB: −2.6 (P = .001) CIP: +2 (P = .05)
rlmI	23S rRNA m5C1962 methyltransferase	Decrease in biofilm formation (Herzberg et al. 2006). Slight growth defect (Purta et al. 2008b).		TOB: −1.4 (NS) CIP: +1.3 (NS)
rlmH N/A	23S rRNA m3Ψ1915 methyltransferase	Slight growth defect (Purta et al. 2008a)		TOB: −3 (P = .003) CIP: −1.3 (NS)
rlmE/rrmJ N/A	23S rRNA 2'-O-ribose U2552 methyltransferase	Decreased growth rate (Caldas et al. 2000, Toh and Mankin 2008, Pletnev et al. 2020). Sparsomycin and tiamulin sensitive (Toh and Mankin 2008). Lincomycin sentitive (Caldas et al. 2000).	Frameshift and stop codon readthrough (Widerak et al. 2005). Accumulation of ribosomal subunit intermediates (Pletnev et al. 2020)	TOB: +1.2 (NS) CIP: +825 (P = .003)
rsmB	16S rRNA m⁵C967 methyltransferase	No obvious growth defect (Gu et al. 1999, Pletnev et al. 2020)	Accumulation of 17S rRNA (Pletnev et al. 2020). Translation initiation (Burakovsky et al. 2012, Arora et al. 2013)	TOB: −6.7 (P = .007) CIP: −2.9 NS
rsmC	16S rRNA m²G1207 methyltransferase	No obvious growth defect (Pletnev et al. 2020)	Correct folding of 16S rRNA (Gc et al. 2020)	TOB: +2.3 (P = .05) CIP: −1.2 (NS)
rsmD	16S rRNA m²G966 methyltransferase	No obvious growth defect (Lesnyak et al. 2007, Pletnev et al. 2020)	Translation initiation (Burakovsky et al. 2012, Arora et al. 2013).	TOB: −5.7 (P = .006) CIP: 1.1 (NS)
rsmF/yebU	16S rRNA m⁵C1407 methyltransferase	No obvious (Pletnev et al. 2020) or slight (Andersen and Douthwaite 2006) growth defect Increased resistance to some AGs reported (Gutierrez et al. 2012)	Role in translation initiation (Das et al. 2008)	TOB: −2.5 (P = .002) CIP: −1.8 (NS)
rsmG N/A	16S rRNA m7G527 methyltransferase	Mutations found in streptomycin resistant MTB clinical isolates (Okamoto et al. 2007)		TOB: −7.7 (P = .0009)
rsmH N/A	16S rRNA m⁴C1402 methyltransferase	No obvious growth defect (Dassain et al. 1999) ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)	Decoding fidelity (Kimura and Suzuki 2010)	TOB: −8.4 (P = .0009)
rsmI N/A	16S rRNA 2'-O-ribose C1402 methyltransferase	No obvious growth defect ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)	Decoding fidelity (Kimura and Suzuki 2010)	TOB: −8.4 (P = 10⁻⁴)
rsuA	16S rRNA pseudouridine516 synthase	No obvious growth defect (Conrad et al. 1999) Overexpression leads to resistance to HOCl (Chen et al. 2021)	Accumulation of 17S rRNA (the present study)	TOB: −2.3 (P = .01) CIP: 1
rluB	23S rRNA pseudouridine2605 synthase	No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008). Increased CM and linezolid sensitivity (Toh and Mankin 2008)	50S subunit maturation (Jiang et al. 2007)	TOB: +9.4 (P = 10⁻⁴) CIP: +1.2 (NS)
rluC N/A	23S rRNA pseudouridine955/2504/2580 synthase	No obvious growth defect (Conrad et al. 1998). Cold sensitivity (Jiang et al. 2007) clindamycin, linezolid, and tiamulin sensitivity (Toh and Mankin 2008)		TOB: +1.8 (P = 10⁻⁴) CIP: +1.3 (NS)
rluD	23S rRNA pseudouridine1911/1915/1917 synthase	Reported to cause a large growth defect in Escherichia coli but independently of pseudourdines (Gutgsell et al. 2001)	Ribosome assembly (Gutgsell et al. 2005)	TOB: +1.4 (P = .03) CIP: +6.6 (P = .002)
rluE	23S rRNA pseudouridine2457 synthase	No obvious growth defect (Del Campo et al. 2001)		TOB: −1.5 (P = .002) CIP: −2.4 (NS)
Modification of both tRNA and rRNA
rluF	23S rRNA pseudouridine2604/tRNATyr pseudouridine35 synthase	No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008, Pletnev et al. 2020). Decreased linezolid resistance (Toh and Mankin 2008)	Effect on translation of Tyr codons (Addepalli and Limbach 2016)	TOB: −1.2 (NS) CIP: −1.2 (NS)
rlmN	tRNA m2A37 methyltransferase/23S rRNA m2A2503 methyltransferase	No obvious growth defect (Benitez-Paez et al. 2012). Slightly increased susceptibility to certain peptidyl transferase-targeting antibiotics (Toh and Mankin 2008)	UAG readthrough (Benitez-Paez et al. 2012)	TOB: +2.3 (P = .005) CIP: −2.2 (P = .0003)

Gene	Function	Known physiological phenotypes in literature	Known effects on translation in literature	TN-seq fold change (T16 antibiotic/T16MH)—this study
tRNA modification
tgt	tRNA-guanine transglycosylase GUN anticodon tRNAs	No significant biological difference but defect in stationary phase viability (Noguchi et al. 1982)	UAG readthrough (Frey et al. 1989). Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: −15.8 (P = .002) CIP: −2 (P = .04)
gidA mnmG	5-carboxymethylamino-methyluridine-tRNA synthase. (m⁵U34)	Pleiotropic phenotypes on DNA replication, cell division, tmRNA tagging defect, reviewed in Nedialkova and Leidel (2015)	Decoding NNG codons (Kurata et al. 2008). Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: +1.9 (NS) CIP: −2.3 (P = .05)
dusA	tRNA-dihydrouridine synthase A	N/A (Bou-Nader et al. 2018)		TOB : +3.8 (P = .0002) CIP : +1.4 (NS)
dusB	tRNA-dihydrouridine synthase B	N/A (Bou-Nader et al. 2018)		TOB : +12.2 (P = 10⁻⁴) CIP : +1.3 (NS)
miaA N/A	tRNA dimethylallyltransferase (i6A37)	Mutator phenotype (Zhao et al. 2001). Reduced tetracycline resistance (Taylor et al. 1998). Evolvability of resistance to CIP (Mehi et al. 2013). Stress specific phenotype: RpoS/IraP translation (Aubee et al. 2016)	Reading frame maintenance defect (Urbonavicius et al. 2001)	TOB: −18 (P = 10⁻⁴)
miaB	Isopentenyl-adenosine A37 tRNA methylthiolase (ms2i6A37) U- starting codons	Stress specific phenotype: fur translation and iron levels (Vecerek et al. 2007)	Taylor et al. (1998)	TOB : −5.9 (P = .0007) CIP: +1.1 (NS)
truA hisT	tRNA pseudouridine38–40 synthase	Growth defect in minimal medium (Tsui et al. 1991)	Reading frame maintenance defect (Urbonavicius et al. 2001). Mistranslation (Bruni et al. 1977, Parker 1982)	TOB: +2.4 (NS) CIP: −2.1 (NS)
truB	tRNA pseudouridine55 synthase	Sensitivity to heatshock (Kinghorn et al. 2002)		TOB: −1.4 (NS) CIP: −1.9 (NS)
truC	tRNA pseudouridine65 synthase tRNAIle1 and tRNAAsp	N/A (Del Campo et al. 2001)		TOB: 1 CIP: 1
trmA	tRNA m⁵U54 methyltransferase and Y341 of tmRNA	N/A (Bjork and Neidhardt 1975). Sensitivity to heatshock of truB trmA (Kinghorn et al. 2002).		TOB: −2.3 (P = .003) CIP: −1.2 (NS)
trmB	tRNA m⁷G46 methyltransferase	N/A (De Bie et al. 2003)		TOB: +2.7 (P = .008) CIP: +3.4 (p = 0.06 NS)
trmEmnmE	5-carboxymethylaminomethyluridine-tRNA synthase m⁵U modification of U34 in tRNA	Pleiotropic, acts with gidA, see gidA above	Reading frame maintenance defect (Urbonavicius et al. 2001). UAG readthrough (Elseviers et al. 1984). Mistranslation (Hagervall et al. 1998)	TOB: +3.9 (P = .01) CIP: +2.4 (NS)
trmH	tRNA (Gm18) 2'-O-methyltransferase	N/A (Persson et al. 1997)		TOB : +5.4 (P = .0002) CIP : −1.3 (NS)
trmK	tRNA (m¹A22)methyltransferase	Kimura et al. (2020)		TOB : 1 CIP : −1.3 (NS)
*rRNA modification (positions described in Escherichia coli)*
rlmB	23S rRNA 2'-O-ribose G2251 methyltransferase	No obvious growth defect (Lovgren and Wikstrom 2001)		TOB: −2.6 (P = .001) CIP: +2 (P = .05)
rlmI	23S rRNA m5C1962 methyltransferase	Decrease in biofilm formation (Herzberg et al. 2006). Slight growth defect (Purta et al. 2008b).		TOB: −1.4 (NS) CIP: +1.3 (NS)
rlmH N/A	23S rRNA m3Ψ1915 methyltransferase	Slight growth defect (Purta et al. 2008a)		TOB: −3 (P = .003) CIP: −1.3 (NS)
rlmE/rrmJ N/A	23S rRNA 2'-O-ribose U2552 methyltransferase	Decreased growth rate (Caldas et al. 2000, Toh and Mankin 2008, Pletnev et al. 2020). Sparsomycin and tiamulin sensitive (Toh and Mankin 2008). Lincomycin sentitive (Caldas et al. 2000).	Frameshift and stop codon readthrough (Widerak et al. 2005). Accumulation of ribosomal subunit intermediates (Pletnev et al. 2020)	TOB: +1.2 (NS) CIP: +825 (P = .003)
rsmB	16S rRNA m⁵C967 methyltransferase	No obvious growth defect (Gu et al. 1999, Pletnev et al. 2020)	Accumulation of 17S rRNA (Pletnev et al. 2020). Translation initiation (Burakovsky et al. 2012, Arora et al. 2013)	TOB: −6.7 (P = .007) CIP: −2.9 NS
rsmC	16S rRNA m²G1207 methyltransferase	No obvious growth defect (Pletnev et al. 2020)	Correct folding of 16S rRNA (Gc et al. 2020)	TOB: +2.3 (P = .05) CIP: −1.2 (NS)
rsmD	16S rRNA m²G966 methyltransferase	No obvious growth defect (Lesnyak et al. 2007, Pletnev et al. 2020)	Translation initiation (Burakovsky et al. 2012, Arora et al. 2013).	TOB: −5.7 (P = .006) CIP: 1.1 (NS)
rsmF/yebU	16S rRNA m⁵C1407 methyltransferase	No obvious (Pletnev et al. 2020) or slight (Andersen and Douthwaite 2006) growth defect Increased resistance to some AGs reported (Gutierrez et al. 2012)	Role in translation initiation (Das et al. 2008)	TOB: −2.5 (P = .002) CIP: −1.8 (NS)
rsmG N/A	16S rRNA m7G527 methyltransferase	Mutations found in streptomycin resistant MTB clinical isolates (Okamoto et al. 2007)		TOB: −7.7 (P = .0009)
rsmH N/A	16S rRNA m⁴C1402 methyltransferase	No obvious growth defect (Dassain et al. 1999) ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)	Decoding fidelity (Kimura and Suzuki 2010)	TOB: −8.4 (P = .0009)
rsmI N/A	16S rRNA 2'-O-ribose C1402 methyltransferase	No obvious growth defect ∆rsmH ∆rsmI has a growth defect (Kimura and Suzuki 2010)	Decoding fidelity (Kimura and Suzuki 2010)	TOB: −8.4 (P = 10⁻⁴)
rsuA	16S rRNA pseudouridine516 synthase	No obvious growth defect (Conrad et al. 1999) Overexpression leads to resistance to HOCl (Chen et al. 2021)	Accumulation of 17S rRNA (the present study)	TOB: −2.3 (P = .01) CIP: 1
rluB	23S rRNA pseudouridine2605 synthase	No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008). Increased CM and linezolid sensitivity (Toh and Mankin 2008)	50S subunit maturation (Jiang et al. 2007)	TOB: +9.4 (P = 10⁻⁴) CIP: +1.2 (NS)
rluC N/A	23S rRNA pseudouridine955/2504/2580 synthase	No obvious growth defect (Conrad et al. 1998). Cold sensitivity (Jiang et al. 2007) clindamycin, linezolid, and tiamulin sensitivity (Toh and Mankin 2008)		TOB: +1.8 (P = 10⁻⁴) CIP: +1.3 (NS)
rluD	23S rRNA pseudouridine1911/1915/1917 synthase	Reported to cause a large growth defect in Escherichia coli but independently of pseudourdines (Gutgsell et al. 2001)	Ribosome assembly (Gutgsell et al. 2005)	TOB: +1.4 (P = .03) CIP: +6.6 (P = .002)
rluE	23S rRNA pseudouridine2457 synthase	No obvious growth defect (Del Campo et al. 2001)		TOB: −1.5 (P = .002) CIP: −2.4 (NS)
Modification of both tRNA and rRNA
rluF	23S rRNA pseudouridine2604/tRNATyr pseudouridine35 synthase	No obvious growth defect (Del Campo et al. 2001, Toh and Mankin 2008, Pletnev et al. 2020). Decreased linezolid resistance (Toh and Mankin 2008)	Effect on translation of Tyr codons (Addepalli and Limbach 2016)	TOB: −1.2 (NS) CIP: −1.2 (NS)
rlmN	tRNA m2A37 methyltransferase/23S rRNA m2A2503 methyltransferase	No obvious growth defect (Benitez-Paez et al. 2012). Slightly increased susceptibility to certain peptidyl transferase-targeting antibiotics (Toh and Mankin 2008)	UAG readthrough (Benitez-Paez et al. 2012)	TOB: +2.3 (P = .005) CIP: −2.2 (P = .0003)

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