**5. rRNA methyltransferases associated with aminoglycoside resistance in** *P. aeruginosa*

Methyltransferases have been intensely studied in *P. aeruginosa*, but this is not the case for RNA methyltransferases, particularly those conferring aminoglycoside resistance. Nowadays, we focus our study in *P. aeruginosa* methyltransferases using

*Pseudomonas aeruginosa - An Armory Within*

in **Figure 2** antibiotic resistance or susceptibility from different *P. aeruginosa* strains

*Strains with P. aeruginosa's genomes showing susceptibility (green) and resistance (red) against some aminoglycosides (amikacin, gentamicin, and tobramycin) and other antipseudomonal antibiotics. Many strains are resistant to a wide range of antibiotics (red with a larger percentage), and most strains are susceptible to* 

Kgm and Kam families are two different groups of SAM-dependent RNA methyltransferases, which modify nucleotides of 16S rRNAs in the specific drug-binding site to confer self-resistance in aminoglycoside-producing bacteria [31]. The Kgm and Kam families have been distinguished based on their nucleotide targets, G1405 and A1408, respectively. The *kgmB* and *armA* genes (Kgm family kanamycin

level of resistance against gentamicin, kanamycin, and tobramycin. The addition of a methyl group in this position interferes directly with the binding to the antibiotic, inducing a steric hindrance between the modified base and the structure of the antibiotic, causing electrostatic repulsions derived from the positive charge in the modified base [32]. On the other hand, the *kamA* and *npmA* genes (Kam family

Another interesting non-aminoglycoside resistance related to RNA methylation is the macrolide-lincosamide-streptogramin-B (MLSB) antibiotics, which is strongly associated with the expression of the methyltransferase of ErmC RNA that causes the dimethylation of the N-6 atom of adenine and interacts with the nucleotide 2058 in the 23S rRNA. Such antibiotics bind to overlap sites within the 50S ribosomal subunit tunnel near the peptidyl transferase center, either by inhibiting the catalysis directly at the peptidyl transferase site or by acting as a physical barrier to the extension of the peptide chain inside the tunnel [33]. Many more *erm*-type methyltransferase genes have been identified in a wide range of Gram-positive and Gram-negative bacteria. Among them, the *ermB*, *ermF*, and *ermA* genes are transferred by transposons, and the *ermC* gene transferred by plasmids. The family of Erm methyltransferases that mediate the mono- or dimethylation of A2058 consists

ring a high level of resistance to kanamycin, apramycin, and neomycin [5].

G1405(N7) position that confers a high

A1408(N1) position confer-

as well as in **Figure 3** with those antibiotics mentioned.

*doxycycline or colistin (green with larger percentage).*

gentamicin methyltransferase) methylate m7

kanamycin-apramycin methyltransferase) methylate m1

of approximately 40 different classes of methylases [34, 35].

**3. Resistance to antibiotics through rRNA methylation**

**56**

**Figure 3.**

molecular biology, genomics, proteomics, chemistry informatics, and bioinformatics [43–45]. RsmG, RsmH, and RsmI are RNA methyltransferases, and these have been broadly studied. Six crystal structures have been reported in PDB for RsmG, from *Thermus thermophilus* with accession numbers 4NXM, 4NXN, 3G88, 3G89 3G8A, and 3G8B [46, 47] and one from *E. coli*, with number 1JSX [48], and another one from *Bacillus subtilis*, with number 1XDZ. RsmH and RsmI crystal structures from *E. coli* are reported in PDB with numbers 3TKA and 5HW4 [49–51]. Checking these three orthologous genes in the PATRIC database, they are being conserved in *P. aeruginosa*'s pan-genome.

RsmG well known as 16S rRNA (guanine527-N7 )-methyltransferase methylates guanine527 at N7 in 16S rRNA [36, 52] (**Table 1**) and catalyzes S-adenosyl-L-methionine + guanine527 in 16S rRNA → S-adenosyl-L-homocysteine + N7 -methylguanine527 in 16S rRNA (see reaction in UniProt, KEGG, or MetaCyc). Researches in *M. tuberculosis* reveal that *rsmG* mutations confer low-level streptomycin resistance; moreover, it has been reported that combining drug resistance mutations of *rsmG* gene remarkably enhances enzyme production in *Paenibacillus agaridevorans* [53]. Likewise, for *P. aeruginosa*, *rsmG* is conserved in both aminoglycoside-resistant and aminoglycoside-susceptible strains.

RsmH also called S-adenosyl-L-methionine (cytosine1402-N4 )-methyltransferase methylates the N4 -of cytosine1402 [54] (**Table 1**). This enzyme catalyzes the following chemical reaction: S-adenosyl-L-methionine + cytosine1402 in 16S rRNA → S-adenosyl-L-homocysteine + N4 -methylcytosine1402 in 16S rRNA (see reaction in UniProt, KEGG, or MetaCyc). Experiments performed with gene knockout of *rsmH* and *rsmI* have shown in *E. coli* BW25113 strain that *ΔrsmH* and *ΔrsmI* increase in doubling times by 15 and 12%, respectively; however, *ΔrsmH/ΔrsmI* increases in doubling time by 29% compared with a wild type cultured at 37°C, indicating that gene knockout caused a slight but significant change in phenotype about cellular growth properties in the absence of both *rsmH* and *rsmI* [54]. As well as *E. coli*, *P. aeruginosa* conserves *rsmH* and *rsmI* genes in both aminoglycosideresistant and susceptible strains; therefore, it is important to study the mutations also in its strains.

RsmI also named S-adenosyl-L-methionine 16S rRNA (cytidine1402–2'-O) methyltransferase methylates in cytidine1402–2'-O (**Table 1**). RsmI catalyzes the next chemical reaction: S-adenosyl-L-methionine + cytidine1402 in 16S rRNA → S-adenosyl-L-homocysteine + 2'-O-methylcytidine1402 (see reaction in UniProt, KEGG, or MetaCyc). RsmI and RsmH react on the same nucleotide, but the first methylates in 2'-O, while the second one in -N4 [54]. Such as *rsmG* and *rsmH*, the *rsmI* gene is also conserved in pan-genome. Theoretical modeling of the structure in RsmI protein from *P. aeruginosa* was performed in iTISSER suit [55], and compared with 5HW4 from *E. coli* (**Figure 4**), the homology and the active site in *P. aeruginosa* are apparently well maintained.

Other interesting *P. aeruginosa* methyltransferases associated with aminoglycoside resistance are m<sup>5</sup> C1404, m1 A1408, and m7 G1405 [6]. Among the last group mentioned, there are some well-studied methyltransferases, such as ArmA, RmtA, RmtB, RmtC, RmtD, RmtF, and RmtG (**Table 2**). This group is characterized for providing resistance to 4,6-disubstituted 2-deoxystreptamine (2-DOS) aminoglycosides [6]. For example, ArmA was found in *Klebsiella pneumoniae* [56]; as for *P. aeruginosa*, among 100 Korean multidrug-resistant isolates, 14 carried this enzyme [57]. The *armA* gene encodes for 16S RNA methyltransferase that methylates guanine (1405)-N<sup>7</sup> . The same gene in *P. aeruginosa* (**Table 2**) presents variable occurrence as it is part of the accessory genome. A multiple alignment, using the listed P. aeruginosa ArmA proteins (16S rRNA (guanine (1405)-N(7))- methyltransferase)) revealed identical homology for this marker.

**59**

The m1

**Table 2.**

**Figure 4.**

*RsmI from P. aeruginosa.*

The location of m7

*belong to India, Brazil, China, and Korea.*

*The Role of Pseudomonas aeruginosa RNA Methyltransferases in Antibiotic Resistance*

G1405 methyltransferase genes across the prokaryotic genome

is variable, as it has been found in several studies mentioned above. The *rmtA* gene in *P. aeruginosa* carries a mobile element Tn5041 [58, 59] identified previously in *Enterobacteriaceae* [56], while the *rmtB* gene identified in *Serratia marcescens* is located in the flanking of Tn3-like region responsible for multiple antimicrobial resistance [59, 60], and both methyltransferases and mobile elements are present in *P. aeruginosa* (**Table 2**). The *rmtC* and *rmtF* genes (**Table 2**) might have been acquired from plasmids as part of mobile genetic elements and finally integrated and stabilized on the chromosome [61]. The *rmtG* gene (**Table 2**) is likely located in the chromosome [62].

*Different 16S rRNA methyltransferases associated with aminoglycoside resistance reported for P. aeruginosa, with classical name, for everyone with the same nucleotide (16S rRNA m7G1405) of isolates from patients who* 

*Structural 3D aligned with chimera 3.1 suit between RsmI proteins from P. aeruginosa (red) and E. coli (blue). Although the alignment displays a structural shifting, the overall topology of the active site is maintained. The protruding residues from each protein depict the active site, harboring SAM (fluorescent green). The structure of E. coli has already been solved by Zhao et al. [49], without obtaining, until now, the crystal structure of* 

**Protein Modification Strain location Reference** ArmA 16S rRNA m7G1405 China and Korea [57, 73] RmtA 16S rRNA m7G1405 Japan and Korea [1, 58, 74, 75] RmtB 16S rRNA m7G1405 China and India [73, 76] RmtC 16S rRNA m7G1405 India [61, 76] RmtD 16S rRNA m7G1405 Brazil [77, 78, 79] RmtF 16S rRNA m7G1405 India [61, 75] RmtG 16S rRNA m7G1405 Brazil [62]

A1408 methyltransferases are present in pan-aminoglycoside-resistant strains,

which were identified by Wachino et al. [63] and provide resistance by these classes of methyltransferases to both 4,5-disubstituted 2-DOS and 4,6-disubstituted 2-DOS aminoglycosides as well as NpmA case. These two classes of methyltransferases are

*DOI: http://dx.doi.org/10.5772/intechopen.85185*

*The Role of Pseudomonas aeruginosa RNA Methyltransferases in Antibiotic Resistance DOI: http://dx.doi.org/10.5772/intechopen.85185*

#### **Figure 4.**

*Pseudomonas aeruginosa - An Armory Within*

*P. aeruginosa*'s pan-genome.

ates guanine527 at N7

methylates the N4

also in its strains.

coside resistance are m<sup>5</sup>

ates guanine (1405)-N<sup>7</sup>

N7

RsmG well known as 16S rRNA (guanine527-N7

coside-resistant and aminoglycoside-susceptible strains.

the first methylates in 2'-O, while the second one in -N4

C1404, m1

transferase)) revealed identical homology for this marker.

in *P. aeruginosa* are apparently well maintained.

rRNA → S-adenosyl-L-homocysteine + N4

RsmH also called S-adenosyl-L-methionine (cytosine1402-N4

following chemical reaction: S-adenosyl-L-methionine + cytosine1402 in 16S

reaction in UniProt, KEGG, or MetaCyc). Experiments performed with gene knockout of *rsmH* and *rsmI* have shown in *E. coli* BW25113 strain that *ΔrsmH* and *ΔrsmI* increase in doubling times by 15 and 12%, respectively; however, *ΔrsmH/ΔrsmI* increases in doubling time by 29% compared with a wild type cultured at 37°C, indicating that gene knockout caused a slight but significant change in phenotype about cellular growth properties in the absence of both *rsmH* and *rsmI* [54]. As well as *E. coli*, *P. aeruginosa* conserves *rsmH* and *rsmI* genes in both aminoglycosideresistant and susceptible strains; therefore, it is important to study the mutations

RsmI also named S-adenosyl-L-methionine 16S rRNA (cytidine1402–2'-O) methyltransferase methylates in cytidine1402–2'-O (**Table 1**). RsmI catalyzes the next chemical reaction: S-adenosyl-L-methionine + cytidine1402 in 16S rRNA → S-adenosyl-L-homocysteine + 2'-O-methylcytidine1402 (see reaction in UniProt, KEGG, or MetaCyc). RsmI and RsmH react on the same nucleotide, but

*rsmH*, the *rsmI* gene is also conserved in pan-genome. Theoretical modeling of the structure in RsmI protein from *P. aeruginosa* was performed in iTISSER suit [55], and compared with 5HW4 from *E. coli* (**Figure 4**), the homology and the active site

Other interesting *P. aeruginosa* methyltransferases associated with aminogly-

A1408, and m7

mentioned, there are some well-studied methyltransferases, such as ArmA, RmtA, RmtB, RmtC, RmtD, RmtF, and RmtG (**Table 2**). This group is characterized for providing resistance to 4,6-disubstituted 2-deoxystreptamine (2-DOS) aminoglycosides [6]. For example, ArmA was found in *Klebsiella pneumoniae* [56]; as for *P. aeruginosa*, among 100 Korean multidrug-resistant isolates, 14 carried this enzyme [57]. The *armA* gene encodes for 16S RNA methyltransferase that methyl-

occurrence as it is part of the accessory genome. A multiple alignment, using the listed P. aeruginosa ArmA proteins (16S rRNA (guanine (1405)-N(7))- methyl-

. The same gene in *P. aeruginosa* (**Table 2**) presents variable

molecular biology, genomics, proteomics, chemistry informatics, and bioinformatics [43–45]. RsmG, RsmH, and RsmI are RNA methyltransferases, and these have been broadly studied. Six crystal structures have been reported in PDB for RsmG, from *Thermus thermophilus* with accession numbers 4NXM, 4NXN, 3G88, 3G89 3G8A, and 3G8B [46, 47] and one from *E. coli*, with number 1JSX [48], and another one from *Bacillus subtilis*, with number 1XDZ. RsmH and RsmI crystal structures from *E. coli* are reported in PDB with numbers 3TKA and 5HW4 [49–51]. Checking these three orthologous genes in the PATRIC database, they are being conserved in

L-methionine + guanine527 in 16S rRNA → S-adenosyl-L-homocysteine +


in 16S rRNA [36, 52] (**Table 1**) and catalyzes S-adenosyl-


)-methyltransferase methyl-


[54]. Such as *rsmG* and

G1405 [6]. Among the last group

)-methyltransferase

**58**

*Structural 3D aligned with chimera 3.1 suit between RsmI proteins from P. aeruginosa (red) and E. coli (blue). Although the alignment displays a structural shifting, the overall topology of the active site is maintained. The protruding residues from each protein depict the active site, harboring SAM (fluorescent green). The structure of E. coli has already been solved by Zhao et al. [49], without obtaining, until now, the crystal structure of RsmI from P. aeruginosa.*


#### **Table 2.**

*Different 16S rRNA methyltransferases associated with aminoglycoside resistance reported for P. aeruginosa, with classical name, for everyone with the same nucleotide (16S rRNA m7G1405) of isolates from patients who belong to India, Brazil, China, and Korea.*

The location of m7 G1405 methyltransferase genes across the prokaryotic genome is variable, as it has been found in several studies mentioned above. The *rmtA* gene in *P. aeruginosa* carries a mobile element Tn5041 [58, 59] identified previously in *Enterobacteriaceae* [56], while the *rmtB* gene identified in *Serratia marcescens* is located in the flanking of Tn3-like region responsible for multiple antimicrobial resistance [59, 60], and both methyltransferases and mobile elements are present in *P. aeruginosa* (**Table 2**). The *rmtC* and *rmtF* genes (**Table 2**) might have been acquired from plasmids as part of mobile genetic elements and finally integrated and stabilized on the chromosome [61]. The *rmtG* gene (**Table 2**) is likely located in the chromosome [62]. The m1 A1408 methyltransferases are present in pan-aminoglycoside-resistant strains, which were identified by Wachino et al. [63] and provide resistance by these classes of methyltransferases to both 4,5-disubstituted 2-DOS and 4,6-disubstituted 2-DOS aminoglycosides as well as NpmA case. These two classes of methyltransferases are

very important for antibiotic resistance, thanks to the similarity of these enzymes with those homologs found in aminoglycoside-producing actinomycetes [6]. Those new genes and proteins will be better studied in expression and structure, to be related to epidemiological data. Looking at **Table 2**, it seems that the expression of methyltransferase might be related to geographical prescription. However, this hypothesis does not seem well founded: microbiological, molecular, and epidemiological understanding of RNA methyltransferases in *P. aeruginosa* will allow the rational use of aminoglycosides and maybe will be not replaced for new antibiotics.

### **6. Final considerations**

Antibiotic resistance is a serious concern for public health and environment. To comprehend the molecular interaction of the methyltransferase in aminoglycoside resistance will be a more efficient way to rationalize its use and consumption. It will be better to clarify the panorama of the rational use of the aminoglycosides to diminish the rapid development of resistance before considering its replacement, since *P. aeruginosa* is still susceptible to them, and, moreover, currently it is known why other *Gammaproteobacteria* are resistant to them. Why are methylation and demethylation a feedback of the antibiotic environmental pressure in bacteria? Bhujbalrao and Anand [64] suggest us some insights using KsgA, exploring the factors which govern the resistance to antibiotics. They observed within loop1 and loop12 of rRNA switched chimera efficiently methylated mini-RNA substrates in vitro, showing that these structural elements suffice for local orientation of the rRNA. In addition, in vivo they notice that the head domain plays a more critical role in leading the enzyme to the select ribosomal region and serves as a sensor of the global environment.

As Kim et al. [65] discuss in letter to editor (Dr. Hur), investigating with P. aeruginosa and aminoglycoside resistance proposes that "less aminoglycoside consumption correlates with less resistance levels"; therefore, we consider that is a requisite for an antibiotic cycling strategy at the global level; also they discussed the rates of amikacin or gentamicin-resistant declining trends, according to the data from KONSAR Korean program in 2011 either for *P. aeruginosa*, *K. pneumoniae*, or *Acinetobacter* spp. [64]. With the knowledge about aminoglycoside resistance molecular mechanisms comparing to the rational prescription cited in Korea, for example, we hypothesize that low methylation rate in the nucleotide substrate of RsmH or RsmI is close to the anchor point of gentamicin in 16S RNA, indicating a possible association with gentamicin or aminoglycosides resistance [5]*.*

RsmG, RsmH, and RsmI methyltransferases belong to the core genome (constitutive genome), while ArmA is part of the accessory genome with identical protein sequences among close species in *Proteobacteria*. Nowadays, the enzymatic activity has been well described; however the antibiotic resistance remains unsolved, perhaps as a consequence of broad usage of aminoglycoside in hospital environment, allowing the development of resistant bacteria. In the future, probably the treatment of *P. aeruginosa* will take into account the genetic trait of each isolate, strain, or species with the set of resistance genes, and surely methyltransferases will be included routinely in clinical care and high throughput or genomic medicine therapies.

### **Acknowledgements**

We thank the University of Antioquia for their financial support to PhD student Jaison H. Cuartas, project CODI 2017-15753.

**61**

**Author details**

Mauricio Corredor\*

Medellín, Colombia

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Pablo Valderrama-Carmona, Jaison H. Cuartas, Diana Carolina Castaño and

Gebiomic group, Natural and Exact Sciences Faculty, University of Antioquia,

\*Address all correspondence to: mauricio.corredor@udea.edu.co

*The Role of Pseudomonas aeruginosa RNA Methyltransferases in Antibiotic Resistance*

*DOI: http://dx.doi.org/10.5772/intechopen.85185*

*The Role of Pseudomonas aeruginosa RNA Methyltransferases in Antibiotic Resistance DOI: http://dx.doi.org/10.5772/intechopen.85185*
