57
In silico identification of auxiliary genes
required for -lactam resistanceVolumen 14 N ú mero 1 - 2 023
ABSTRACT
In silico identification of auxiliary genes required for
β-lactam resistance
(1) Carrera de Nutrición y Dietética. Escuela Superior Politécnica de Chimborazo, Riobamba, 060101, Ecuador.
* Correspondence to: igor.astudillo@espoch.edu.ec
Igor Eduardo Astudillo Skliarova ¹ igor.astudillo@espoch.edu.ec
Staphylococcus aureus is a type of bacteria commonly found on the skin and in the nasal passages of healthy
individuals. However, it can also cause a range of infections in clinical settings. One of the most concerning aspects
of S. aureus is its ability to develop antibiotic resistance. Methicillin-resistant S. aureus (MRSA) is a strain of the
bacteria that is resistant to many antibiotics and can be difficult to treat. The primary mechanism of methicillin
resistance in MRSA is the presence of the mecA gene, which encodes for a modified penicillin-binding protein
known as PBP2a. This protein has a lower affinity for beta-lactam antibiotics. Another gene, blaZ, is also present
in MRSA and encodes for a beta-lactamase enzyme that can hydrolyse and inactivate beta-lactam antibiotics
such as penicillin. In addition, there are several auxiliary factors that can contribute to beta-lactam resistance.
They can include efflux pumps, enzymes that modify or degrade antibiotics, and bacterial cell wall modifications
that reduce the affinity of antibiotics for their targets. In this study, with the aid of the in silico identification
method, we identify the novel auxiliary factors aux1, aux2, aux4, aux11, aux14, aux16 and aux19. Next, we show
that aux2, aux4, aux11, aux14 are not directly involved in β-lactam resistance, but may contribute through other
mechanisms that decrease the efficacy of these antibiotics, whereas aux16 and aux19 are directly associated
with β-lactam and bacitracin resistance, respectively. Understanding the various auxiliary factors that contribute
to beta-lactam resistance can help guide the development of new antibiotics and other therapeutic strategies.
Keywords: auxiliary factors, Staphylococcus aureus, β-lactam antibiotic, antibiotic resistance, in silico
identification..
Facultad de
Salud Pública
Facultad de
Salud Pública
CSSNLa Ciencia al Servicio de la Salud y la Nutrición
REVISTA CIENTÍFICA DIGITAL
La Ciencia al Servicio de la Salud y la Nutrición
http://revistas.espoch.edu.ec/index.php/cssn
ISSN 1390-874X BY
ARTÍCULOS ORIGINALES
1. INTRODUCTION
Staphylococcus aureus is a Gram-positive bacterium
that is commonly found on the skin and mucous
membranes of humans and other animals. It is a
leading cause of healthcare-associated infections,
including bacteremia, sepsis, toxic shock syndrome,
and skin and soft tissue infections (1).Staphylococcus
aureus is able to acquire resistance to multiple
antibiotics, including methicillin, through the
acquisition of antibiotic resistance genes. This ability
to resist antibiotics has made it difficult to treat S.
aureus infections and has led to the emergence
of methicillin-resistant S. aureus (MRSA). MRSA is
highly transmissible and can spread through contact
with infected persons or contaminated surfaces
and, therefore, it poses a major challenge to public
health and medical care (2).
Methicillin-resistant Staphylococcus aureus (MRSA)
is classified into three main types: healthcare-
associated (HA-MRSA), community-associated
(CA-MRSA) and livestock-associated (LA-MRSA)
(3). CA-MRSA strains are usually acquired in the
community and typically causes mild to moderate
skin and soft tissue infections, such as boils
and abscesses. One of the most important and
common CA-MRSA strains is MW2 (4).
MRSA strains possess the mecA gene, which
encodes the alternative penicillin-binding protein
PBP2A. This protein confers resistance to β-lactam
antibiotics by decreasing their ability to bind to
the cell wall. Additionally, some MRSA strains may
encode the beta-lactamase BlaZ, which hydrolyses
β-lactam antibiotics (5).
Although the presence of mecA is necessary
for the resistance to β-lactam antibiotics, it is
not sufficient. Therefore, apart from mecA and
blaZ, many other genes involved in resistance to
β-lactam antibiotics have been identified and
58Igor Eduardo Astudillo Skliarova
http://revistas.espoch.edu.ec/index.php/cssn
named such as fem genes (factors essential for
methicillin resistance) and aux genes (auxiliary
genes). Both of these factors are not related to
mecA gene (6). A list of currently known accessory
and auxiliary factors is presented on Table 1.
Auxiliary factors may affect the ability of
antibiotics to penetrate the bacterial cell wall or
alter the expression of resistance genes (28). One
such factor is the presence of efflux pumps, which
are membrane proteins that can pump antibiotics
out of the bacterial cell. This can reduce the
concentration of antibiotics inside the cell and
make it more difficult for the antibiotics to be
effective (6). Another factor is the presence of
enzymes that can modify or degrade antibiotics.
Table 1: List of auxiliary factors.
For example, some bacteria produce beta-
lactamases that can hydrolyse and inactivate
beta-lactam antibiotics (29). Bacterial cell wall
modifications, such as the addition of amino acids
or sugars to the peptidoglycan layer, can also
contribute to beta-lactam resistance by reducing
the affinity of antibiotics for their targets (30).
Although the mechanisms of resistance to β-lactam
used by S. aureus have been studied for many
years, there are still many unclear mechanisms.
Therefore, the aim of the present study is to
identify novel putative auxiliary factors genes
present in S. aureus using the in silico identification
method.
Gene Function
glmM Phosphoglucosamine mutase: conversion of glucosamine-6-phosphate to glucosamine-1-phosphate dur-
ing the early stage of peptidoglycan synthesis (7).
glmS Glucosamine-fructose-6-phosphate aminotransferase: conversion of D-fructose-6-phosphate to D-glu-
cosamine 6-phosphate (8).
murA Transferase: conversion of UDP-GlcNAc to UDP-GlcNAc-enoylpyruvate (9).
murB Reductase: conversion of UDP-GlcNAc-enoylpyruvate to UDP-MurNAc (9).
murC-F Mur ligases: sequential addition of the aminoacids L-Ala, D-Glu, L-Lys, and D-Ala-D-Ala to build the tetra-
peptide side chain (10).
glyS Glycine tRNA synthetase: provides a glycine substrate for the assembly of the pentaglycine bridge (11).
femX Peptidyltransferase: Incorporation of the first glycine of the pentaglycine bridge (12).
femA Peptidyltransferase: Incorporation of the glycines 2 and 3 of the pentaglycine bridge (12).
femB Peptidyltransferase: Incorporation of the glycines 4 and 5 of the pentaglycine bridge (12).
femC Glutamine synthetase repressor: Participates in the tetrapeptide side chain amidation (13).
gatD/murT Amidotransferase complex: amidation of lipid II (14).
SAV1754 Probably functions as a lipid II flippase during its translocation to the outer leaflet of the cell membrane (15).
ftsW Proposed lipid II flippase (16).
pbp1 PBP, possesses transpeptidase activity (17).
pbp2 Bifunctional PBP with transglycosylase and transpeptidase domains (17).
pbp4 PBP, possesses transpeptidase and carboxipeptidase activities (18).
prsA Probably required for posttranscriptional maturation of PBP2a (19).
fmtA Accessory PBP, possesses transpeptidase activity and has low affinity to penicillin (20).
Llm (tarO) Transferase: transfer of an N-acetylglucosamine-1-phosphate residue from UDP-N-acetylglucosamine to
undecaprenyl-phosphate to produce the WTA intermediate lipid α (21).
tarA Transferase: transfer of an N-acetylmannosamine residue to the lipid α to form the intermediate lipid β (21).
tarB Transferase: transfer of an sn-glycerol-3-phosphate unit to generate the intermediate lipid ϕ.1 (22).
tarD Cytidylyltransferase: production of CDP-glycerol for TarB during WTA synthesis (22).
tarL Cytidylyltransferase: required for ribitol phosphate polymerisation of WTA (22).
tarI CDP-ribitol synthase: Synthesis of CDP-ribitol for TarL during WTA synthesis (22).
tarS
ltaS
Glycosyltransferase: Addition of β-O-GlcNAc to the WTA (22).
LTA synthesis (23).
spsB Signal peptidase enzyme: posttranscriptional regulation of the protein LtaS (24).
pknB Eukaryote-like serine/threonine kinases: Associated with increased resistance to β-lactam antibiotics (25).
sigB Alternative sigma factor: Involved in β-lactam resistance (26).
vraSR Two-component regulatory system: activation of specific genes required for antibiotic resistance in re-
sponse to cell wall stress (27).
http://revistas.espoch.edu.ec/index.php/cssn
named such as fem genes (factors essential for
methicillin resistance) and aux genes (auxiliary
genes). Both of these factors are not related to
mecA gene (6). A list of currently known accessory
and auxiliary factors is presented on Table 1.
Auxiliary factors may affect the ability of
antibiotics to penetrate the bacterial cell wall or
alter the expression of resistance genes (28). One
such factor is the presence of efflux pumps, which
are membrane proteins that can pump antibiotics
out of the bacterial cell. This can reduce the
concentration of antibiotics inside the cell and
make it more difficult for the antibiotics to be
effective (6). Another factor is the presence of
enzymes that can modify or degrade antibiotics.
Table 1: List of auxiliary factors.
For example, some bacteria produce beta-
lactamases that can hydrolyse and inactivate
beta-lactam antibiotics (29). Bacterial cell wall
modifications, such as the addition of amino acids
or sugars to the peptidoglycan layer, can also
contribute to beta-lactam resistance by reducing
the affinity of antibiotics for their targets (30).
Although the mechanisms of resistance to β-lactam
used by S. aureus have been studied for many
years, there are still many unclear mechanisms.
Therefore, the aim of the present study is to
identify novel putative auxiliary factors genes
present in S. aureus using the in silico identification
method.
Gene Function
glmM Phosphoglucosamine mutase: conversion of glucosamine-6-phosphate to glucosamine-1-phosphate dur-
ing the early stage of peptidoglycan synthesis (7).
glmS Glucosamine-fructose-6-phosphate aminotransferase: conversion of D-fructose-6-phosphate to D-glu-
cosamine 6-phosphate (8).
murA Transferase: conversion of UDP-GlcNAc to UDP-GlcNAc-enoylpyruvate (9).
murB Reductase: conversion of UDP-GlcNAc-enoylpyruvate to UDP-MurNAc (9).
murC-F Mur ligases: sequential addition of the aminoacids L-Ala, D-Glu, L-Lys, and D-Ala-D-Ala to build the tetra-
peptide side chain (10).
glyS Glycine tRNA synthetase: provides a glycine substrate for the assembly of the pentaglycine bridge (11).
femX Peptidyltransferase: Incorporation of the first glycine of the pentaglycine bridge (12).
femA Peptidyltransferase: Incorporation of the glycines 2 and 3 of the pentaglycine bridge (12).
femB Peptidyltransferase: Incorporation of the glycines 4 and 5 of the pentaglycine bridge (12).
femC Glutamine synthetase repressor: Participates in the tetrapeptide side chain amidation (13).
gatD/murT Amidotransferase complex: amidation of lipid II (14).
SAV1754 Probably functions as a lipid II flippase during its translocation to the outer leaflet of the cell membrane (15).
ftsW Proposed lipid II flippase (16).
pbp1 PBP, possesses transpeptidase activity (17).
pbp2 Bifunctional PBP with transglycosylase and transpeptidase domains (17).
pbp4 PBP, possesses transpeptidase and carboxipeptidase activities (18).
prsA Probably required for posttranscriptional maturation of PBP2a (19).
fmtA Accessory PBP, possesses transpeptidase activity and has low affinity to penicillin (20).
Llm (tarO) Transferase: transfer of an N-acetylglucosamine-1-phosphate residue from UDP-N-acetylglucosamine to
undecaprenyl-phosphate to produce the WTA intermediate lipid α (21).
tarA Transferase: transfer of an N-acetylmannosamine residue to the lipid α to form the intermediate lipid β (21).
tarB Transferase: transfer of an sn-glycerol-3-phosphate unit to generate the intermediate lipid ϕ.1 (22).
tarD Cytidylyltransferase: production of CDP-glycerol for TarB during WTA synthesis (22).
tarL Cytidylyltransferase: required for ribitol phosphate polymerisation of WTA (22).
tarI CDP-ribitol synthase: Synthesis of CDP-ribitol for TarL during WTA synthesis (22).
tarS
ltaS
Glycosyltransferase: Addition of β-O-GlcNAc to the WTA (22).
LTA synthesis (23).
spsB Signal peptidase enzyme: posttranscriptional regulation of the protein LtaS (24).
pknB Eukaryote-like serine/threonine kinases: Associated with increased resistance to β-lactam antibiotics (25).
sigB Alternative sigma factor: Involved in β-lactam resistance (26).
vraSR Two-component regulatory system: activation of specific genes required for antibiotic resistance in re-
sponse to cell wall stress (27).
59
In silico identification of auxiliary genes
required for -lactam resistanceVolumen 14 N ú mero 1 - 2 023
2. METHODOLOGY
In silico gene identification
The sequence of the genes of interest was
downloaded from the nucleotide databases of the
National Center for Biotechnology Information
(NCBI) (http://www.ncbi.nlm.nih.gov/) and the
Kyoto Encyclopedia of Genes and Genomes
(KEGG) (https://www.genome.jp/kegg/).
In silico protein characterisation
With the aid of the programme protein-protein
BLAST (Basic Local Alignment Search Tool) (http://
blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins),
the translated sequence of the genes was aligned
with the sequence of amino acids of already
known and studied proteins in order to predict
the function of the protein encoded by the gene
of interest. Several characteristics of the protein
such as the isoelectric point (pI), molecular
weight (mW), co-localisation with other genes,
the transmembrane helices, homology or
structure were predicted with the aid of the
online available programmes: Expert Protein
Analysis System (https://www.expasy.org/),
DeepTMHMM (http://www.cbs.dtu.dk/services/
TMHMM/) and Homology detection & structure
prediction by HMM-HMM comparison (HHpred)
(https://toolkit.tuebingen.mpg.de/tools/hhpred).
3. RESULTS
In 1999 the identification of 21 novel auxiliary
genes was reported in the Microbial Drug
Resistance journal (31). Unfortunately, after
this, some of these genes have not been further
characterised and the function of many of these
auxiliary genes is still unknown. In this study, we
decided to predict the function and properties of
the proteins encoded by uncharacterised genes
by using different bioinformatics tools.
The sequence data of the aux genes: aux1,
aux2, aux4, aux11, aux14, aux16 and aux19, was
retrieved with the aid of NCBI using the accession
numbers found in the afore-mentioned report.
As these genes were discovered in S. aureus strain
COL. In order to predict the preliminary name
of the gene and the function of its product, the
amino acid sequence was aligned with homolog
sequences of other organisms using protein-
protein BLAST. This and other information related
to the protein were collected with the aid of the
following online available softwares: ExPaSy,
TMHMM Server v. 2.0 and HHpred. All the data
is shown in Table 2. Our results suggest that,
among all the genes described by De Lencastre
et al. in 1999 (31), aux16 and aux19 were directly
associated with antibiotic resistance.
Table 2: Characteristics of new auxiliary genes generated in the background of S. aureus strain MW2.
ND, not determined; ¹Partial sequence analysis; ²Metallo β-lactamase.
aux
gene
Accession
number ORF Putative
gene
Length
(aa) pI/MW Stoichiometry Solubility Encoded protein Function
aux1 Y18639 1 prpC 247 5.10 / 28076 Monomer Soluble Phosphorylated protein
phosphatase Cellular regulation
aux2 Y13639 388 pknB 664 5.76 / 74363 Monomer Trans-
membrane Protein kinase Cellular regulation
aux4 Y18630 - ccpA 329 5.58 / 36060 Monomer Soluble Catabolite Control Protein A Transcriptional regulation
aux11 Y18632 - lysA 421 5.58 / 47035 Homodimer Soluble diaminopimelate decar-
boxylase L-lysine biosynthesis
aux14 Y14324 271 - 303 5.64 / 34812 ND Soluble RNase adapter protein RapZ Cellular regulation
aux16 AJ131754
1 1 cutD 540 8.93 / 60254 Homotrimer Trans-
membrane Osmoprotectant transporter Choline and betaine/carnitine
transporter
2 blaMBL
2 228 5.68 / 26239 Monomer Soluble MBL2 Resistance to broad range of
β-lactam resistance
4 gbsA 496 4.96 / 54623 Homotetra-
mer Soluble Betaine aldehyde dehydro-
genase
Glycine, serine and threonine
metabolism. Betaine biosynthesis
via choline pathway
5 1 betA 569 7.08 / 63624 Monomer Soluble Choline dehydrogenase
Glycine, serine and threonine
metabolism. Oxidation of choline
to betaine aldehyde and betaine
aldehyde to glycine betaine
aux19 Y18641
1 vraD 252 7.03 / 27775 Monomer Soluble Membrane transport and
signal transduction
ABC transporter. Also part of a
two component system. Associa-
ted to bacitracin
2
vraE
626 9.65 / 70105 Homotrimer Trans-
membrane
Membrane transport and
signal transduction
ABC transporter. Also part of a
two component system. Associa-
ted to bacitracin resistance
In silico identification of auxiliary genes
required for -lactam resistanceVolumen 14 N ú mero 1 - 2 023
2. METHODOLOGY
In silico gene identification
The sequence of the genes of interest was
downloaded from the nucleotide databases of the
National Center for Biotechnology Information
(NCBI) (http://www.ncbi.nlm.nih.gov/) and the
Kyoto Encyclopedia of Genes and Genomes
(KEGG) (https://www.genome.jp/kegg/).
In silico protein characterisation
With the aid of the programme protein-protein
BLAST (Basic Local Alignment Search Tool) (http://
blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins),
the translated sequence of the genes was aligned
with the sequence of amino acids of already
known and studied proteins in order to predict
the function of the protein encoded by the gene
of interest. Several characteristics of the protein
such as the isoelectric point (pI), molecular
weight (mW), co-localisation with other genes,
the transmembrane helices, homology or
structure were predicted with the aid of the
online available programmes: Expert Protein
Analysis System (https://www.expasy.org/),
DeepTMHMM (http://www.cbs.dtu.dk/services/
TMHMM/) and Homology detection & structure
prediction by HMM-HMM comparison (HHpred)
(https://toolkit.tuebingen.mpg.de/tools/hhpred).
3. RESULTS
In 1999 the identification of 21 novel auxiliary
genes was reported in the Microbial Drug
Resistance journal (31). Unfortunately, after
this, some of these genes have not been further
characterised and the function of many of these
auxiliary genes is still unknown. In this study, we
decided to predict the function and properties of
the proteins encoded by uncharacterised genes
by using different bioinformatics tools.
The sequence data of the aux genes: aux1,
aux2, aux4, aux11, aux14, aux16 and aux19, was
retrieved with the aid of NCBI using the accession
numbers found in the afore-mentioned report.
As these genes were discovered in S. aureus strain
COL. In order to predict the preliminary name
of the gene and the function of its product, the
amino acid sequence was aligned with homolog
sequences of other organisms using protein-
protein BLAST. This and other information related
to the protein were collected with the aid of the
following online available softwares: ExPaSy,
TMHMM Server v. 2.0 and HHpred. All the data
is shown in Table 2. Our results suggest that,
among all the genes described by De Lencastre
et al. in 1999 (31), aux16 and aux19 were directly
associated with antibiotic resistance.
Table 2: Characteristics of new auxiliary genes generated in the background of S. aureus strain MW2.
ND, not determined; ¹Partial sequence analysis; ²Metallo β-lactamase.
aux
gene
Accession
number ORF Putative
gene
Length
(aa) pI/MW Stoichiometry Solubility Encoded protein Function
aux1 Y18639 1 prpC 247 5.10 / 28076 Monomer Soluble Phosphorylated protein
phosphatase Cellular regulation
aux2 Y13639 388 pknB 664 5.76 / 74363 Monomer Trans-
membrane Protein kinase Cellular regulation
aux4 Y18630 - ccpA 329 5.58 / 36060 Monomer Soluble Catabolite Control Protein A Transcriptional regulation
aux11 Y18632 - lysA 421 5.58 / 47035 Homodimer Soluble diaminopimelate decar-
boxylase L-lysine biosynthesis
aux14 Y14324 271 - 303 5.64 / 34812 ND Soluble RNase adapter protein RapZ Cellular regulation
aux16 AJ131754
1 1 cutD 540 8.93 / 60254 Homotrimer Trans-
membrane Osmoprotectant transporter Choline and betaine/carnitine
transporter
2 blaMBL
2 228 5.68 / 26239 Monomer Soluble MBL2 Resistance to broad range of
β-lactam resistance
4 gbsA 496 4.96 / 54623 Homotetra-
mer Soluble Betaine aldehyde dehydro-
genase
Glycine, serine and threonine
metabolism. Betaine biosynthesis
via choline pathway
5 1 betA 569 7.08 / 63624 Monomer Soluble Choline dehydrogenase
Glycine, serine and threonine
metabolism. Oxidation of choline
to betaine aldehyde and betaine
aldehyde to glycine betaine
aux19 Y18641
1 vraD 252 7.03 / 27775 Monomer Soluble Membrane transport and
signal transduction
ABC transporter. Also part of a
two component system. Associa-
ted to bacitracin
2
vraE
626 9.65 / 70105 Homotrimer Trans-
membrane
Membrane transport and
signal transduction
ABC transporter. Also part of a
two component system. Associa-
ted to bacitracin resistance
60Igor Eduardo Astudillo Skliarova
http://revistas.espoch.edu.ec/index.php/cssn
This suggests that all the three genes are involved in cellular regulation. Moreover, a DeepTMHMM
analysis shows that aux2 is a transmembrane protein (Figure 1). The gene aux4 matched the catabolite
control protein a with 99.66% probability (Figure 1), which suggests that this gene is involved in
transcriptional regulation. Finally, the gene aux11 matched the diaminopimelate decarboxylase gene
with 100% probability (Figure 1). Diaminopimelate decarboxylase is involved in L-lysine biosynthesis
(32).
The DNA sequences aux1, aux2, aux4 and aux14 are associated with regulation mechanisms,
whereas aux11 is associated with biosynthetic pathways.
The genes aux1, aux2 and aux14 matched respectively the phosphorylated protein phosphatase with
100% probability, the protein kinase with 100% probability and RNase adapter protein RapZ with 100%
probability (Figure 1).
a
c
e
b
d
f
Figure 1: HHpred analysis of putative auxiliary factors aux1 (a), aux2 (b), aux4 (c), aux11 (d) and aux14 (e), and DeepTMHMM
analysis of aux2, showing the presence of a membrane-bound domain.
http://revistas.espoch.edu.ec/index.php/cssn
This suggests that all the three genes are involved in cellular regulation. Moreover, a DeepTMHMM
analysis shows that aux2 is a transmembrane protein (Figure 1). The gene aux4 matched the catabolite
control protein a with 99.66% probability (Figure 1), which suggests that this gene is involved in
transcriptional regulation. Finally, the gene aux11 matched the diaminopimelate decarboxylase gene
with 100% probability (Figure 1). Diaminopimelate decarboxylase is involved in L-lysine biosynthesis
(32).
The DNA sequences aux1, aux2, aux4 and aux14 are associated with regulation mechanisms,
whereas aux11 is associated with biosynthetic pathways.
The genes aux1, aux2 and aux14 matched respectively the phosphorylated protein phosphatase with
100% probability, the protein kinase with 100% probability and RNase adapter protein RapZ with 100%
probability (Figure 1).
a
c
e
b
d
f
Figure 1: HHpred analysis of putative auxiliary factors aux1 (a), aux2 (b), aux4 (c), aux11 (d) and aux14 (e), and DeepTMHMM
analysis of aux2, showing the presence of a membrane-bound domain.
61
In silico identification of auxiliary genes
required for -lactam resistanceVolumen 14 N ú mero 1 - 2 023
The DNA sequence aux19 was not directly associated with β-lactam resistance, but was associated
with bacitracin resistance.
The DNA sequence of aux19 comprises two ORFs. These ORFs were predicted to act as ABC transporters
and be part of a two component system (Table 2)(Figure 3). Although these ORFs were not predicted
a
c
b
d
e
Figure 2: HHpred analysis of the putative proteins associated with the putative auxiliary factor aux16: cutD (a), blaMBL
(b), gbsA (c) and betA (d), and DeepTMHMM analysis of putative protein cutD, showing the presence of membrane-bound
domains.
The DNA sequence aux16 could be associated with β-lactam resistance
The DNA sequence aux16 comprises 5 open reading frames (ORFs). Among these ORFs, the ORF2
was predicted with 99.85% probability to belong to the Subclass B3 metallo-beta-lactamases (MBL)
(Figure 2), which are a type of bacterial enzymes that can inactivate many beta-lactam antibiotics,
such as penicillins, cephalosporins, and carbapenems (33). Additionally, other ORFs associated with
aux16 may be involved in glycine, serine and threonine metabolism, betaine biosynthesis via choline
pathway and oxidation of choline to betaine aldehyde and betaine aldehyde to glycine betaine (Table
2). Interestingly, the ORF1 of aux16 was predicted to be a membrane-anchored protein (Figure 2) and
act as a Choline and betaine/carnitine transporter.
In silico identification of auxiliary genes
required for -lactam resistanceVolumen 14 N ú mero 1 - 2 023
The DNA sequence aux19 was not directly associated with β-lactam resistance, but was associated
with bacitracin resistance.
The DNA sequence of aux19 comprises two ORFs. These ORFs were predicted to act as ABC transporters
and be part of a two component system (Table 2)(Figure 3). Although these ORFs were not predicted
a
c
b
d
e
Figure 2: HHpred analysis of the putative proteins associated with the putative auxiliary factor aux16: cutD (a), blaMBL
(b), gbsA (c) and betA (d), and DeepTMHMM analysis of putative protein cutD, showing the presence of membrane-bound
domains.
The DNA sequence aux16 could be associated with β-lactam resistance
The DNA sequence aux16 comprises 5 open reading frames (ORFs). Among these ORFs, the ORF2
was predicted with 99.85% probability to belong to the Subclass B3 metallo-beta-lactamases (MBL)
(Figure 2), which are a type of bacterial enzymes that can inactivate many beta-lactam antibiotics,
such as penicillins, cephalosporins, and carbapenems (33). Additionally, other ORFs associated with
aux16 may be involved in glycine, serine and threonine metabolism, betaine biosynthesis via choline
pathway and oxidation of choline to betaine aldehyde and betaine aldehyde to glycine betaine (Table
2). Interestingly, the ORF1 of aux16 was predicted to be a membrane-anchored protein (Figure 2) and
act as a Choline and betaine/carnitine transporter.
62Igor Eduardo Astudillo Skliarova
http://revistas.espoch.edu.ec/index.php/cssn
4. DISCUSSIONS
As already mentioned, auxiliary factors
significantly contribute to β-lactam resistance
and during the last decades many of them have
been described. These factors have been studied
with the aid of transposon mutagenesis using
the transposon (Tn551) (35). In 1994, a large
Tn551 library produced in the background of S.
aureus strain COL was screened for mutants with
decreased levels of resistance to methicillin. The
complete DNA sequence of the 21 new auxiliary
genes derived from this library was published in
1999 (31).
to be directly associated with β-lactam resistance (Table 2)(Figure 3), they were predicted to be
associated with resistance to bacitracin, which is an antibiotic that inhibits the synthesis of the cell
wall (34).
The DNA sequence aux19 was not directly associated with β-lactam resistance, but was associated
with bacitracin resistance.
The DNA sequence of aux19 comprises two ORFs. These ORFs were predicted to act as ABC transporters
and be part of a two component system (Table 2)(Figure 3). Although these ORFs were not predicted
to be directly associated with β-lactam resistance (Table 2)(Figure 3), they were predicted to be
associated with resistance to bacitracin, which is an antibiotic that inhibits the synthesis of the cell
wall (34).
a b
c
Figure 3: HHpred analysis of the putative proteins associated with the putative auxiliary factor aux19: vraD (a) and vraE (b),
and DeepTMHMM analysis of putative protein vraE, showing the presence of membrane-bound domains.
In this study, seven of the 21 new auxiliary genes
were characterised by in silico sequence analysis.
As we can observe in Table 9, most of the genes
are involved in metabolic pathways, transport
and regulation of gene expression.
It is known that any kind of selective pressure,
including antibiotic exposure, causes dramatic
changes within the cell and this implies additional
metabolic costs. Therefore, in order to withstand
the damage caused by cell wall targeting
antibiotics, the cell must produce additional
http://revistas.espoch.edu.ec/index.php/cssn
4. DISCUSSIONS
As already mentioned, auxiliary factors
significantly contribute to β-lactam resistance
and during the last decades many of them have
been described. These factors have been studied
with the aid of transposon mutagenesis using
the transposon (Tn551) (35). In 1994, a large
Tn551 library produced in the background of S.
aureus strain COL was screened for mutants with
decreased levels of resistance to methicillin. The
complete DNA sequence of the 21 new auxiliary
genes derived from this library was published in
1999 (31).
to be directly associated with β-lactam resistance (Table 2)(Figure 3), they were predicted to be
associated with resistance to bacitracin, which is an antibiotic that inhibits the synthesis of the cell
wall (34).
The DNA sequence aux19 was not directly associated with β-lactam resistance, but was associated
with bacitracin resistance.
The DNA sequence of aux19 comprises two ORFs. These ORFs were predicted to act as ABC transporters
and be part of a two component system (Table 2)(Figure 3). Although these ORFs were not predicted
to be directly associated with β-lactam resistance (Table 2)(Figure 3), they were predicted to be
associated with resistance to bacitracin, which is an antibiotic that inhibits the synthesis of the cell
wall (34).
a b
c
Figure 3: HHpred analysis of the putative proteins associated with the putative auxiliary factor aux19: vraD (a) and vraE (b),
and DeepTMHMM analysis of putative protein vraE, showing the presence of membrane-bound domains.
In this study, seven of the 21 new auxiliary genes
were characterised by in silico sequence analysis.
As we can observe in Table 9, most of the genes
are involved in metabolic pathways, transport
and regulation of gene expression.
It is known that any kind of selective pressure,
including antibiotic exposure, causes dramatic
changes within the cell and this implies additional
metabolic costs. Therefore, in order to withstand
the damage caused by cell wall targeting
antibiotics, the cell must produce additional
63
In silico identification of auxiliary genes
required for -lactam resistanceVolumen 14 N ú mero 1 - 2 023
proteins required for cell wall maintenance (36).
Notably, one of the aux genes – aux11 has high
homology with the gene lysA, which encodes
diaminopimelate decarboxylase. This protein
is required for the synthesis of the amino acid
L-lysine (32), which is part of the tetrapeptide
side chain of the PG (37). In consequence, it is
possible to speculate that, in the presence of
β-lactam antibiotics, enhanced expression of
diaminopimelate decarboxylase is crucial to
the cross-linking of PG, performed by penicillin
binding proteins.
When resistant bacteria are exposed to antibiotics,
before they synthesise antibiotic resistance
proteins such as PBP2a, they temporarily become
more vulnerable to osmotic stress. Therefore, the
bacterial cell must activate genes required for
the transport or synthesis of osmoprotectants
or compatible solutes, which contribute to
withstanding osmotic stress (38). Some putative
genes that might play that important role have
been identified in this study: cutD, gbsA and
betA. All these genes were initially identified as
aux16. The putative gene cutD was predicted to
have transmembrane domains, which correlates
with the predicted function of its product. The
gene cutD may be required for the transport of
osmoprectants such as betaine or precursors
of other osmoprotectants such as choline (39).
The putative genes gbsA and betA encode the
proteins: betaine aldehyde dehydrogenase
and choline dehydrogenase, respectively.
Betaine aldehyde dehydrogenase mediates the
biosynthesis of the osmoprotectant betaine,
whereas choline dehydrogenase is responsible
for the synthesis of the osmoprotectant glycine
betaine (40).
β-lactam antibiotics induce the expression of
operons that contain β-lactam resistance genes.
Furthermore, there is evidence that β-lactam
antibiotics also trigger a global stress response in
bacterial cells. This occurs when the cell detects
cell-wall synthesis perturbations with the aid of
the specific VraS/R two-component system (27).
In this study we identified two putative genes:
vraD and vraE (initially identified as aux19), whose
products might be part of VraS/R two-component
system. The activation of this two-component
system leads to massive overexpression of genes,
many of which are crucial for β-lactam antibiotic
resistance (27). For instance, VraS/R induces the
expression of RelP and RelQ, which synthesises
alarmones during the exponential phase of
growth (41). Alarmones are generally produced
during nutrient starvation (for instance, amino
acid starvation) and play a principal role in
stringent response, whereby the synthesis of
rRNA, tRNA and amino acids available in the
cell is repressed and synthesis of amino acids
present in insufficient amounts within the cell
is induced (42). The principal known alarmones
are guanosine tetraphosphate (ppGpp) and
guanosine pentaphosphate (pppGpp). During
nutrient starvation, these alarmones are
produced by specific proteins such as RelA or
SpoT, which become activated when uncharged
tRNA molecules bind the ribosomal site A (43).
There is evidence that this alarmones increase
the resistance of MRSA to antibiotics (44).
The global stress response requires the
participation of many different genes involved
in cascade reactions such as protein kinases,
phosphatases or transcriptional regulators.
These molecules play similar roles in prokaryotes
and eukaryotes (45). We identified three putative
genes whose products may participate in this
cascade: prpC, pknB and ccpA (initially identified
as aux1, aux2 and aux4, respectively). We can
presume that the products of these genes
may also be involved in the cascade reactions
triggered by the VraS/R two-component system,
but this proposed linkage must be verified
experimentally.
5. CONCLUSIONS
In conclusion, we can confirm that the gene
blrA is required for β-lactam resistance in the
CA-MRSA strain MW2, but does not affect the
expression of the gene mecA or the structure of
the wall teichoic acids. At the same time, many
auxiliary genes present in CA-MRSA strain MW2
may be involved in global cellular regulation,
stress response and metabolic pathways.
The author declares no conflict of interest
The author takes complete responsibility for the
information presented in this original scientific
article
6. CONFLICT OF INTEREST
7. LIMITATIONS OF LIABILITY
The present Research Project was funded by
Secretaría Nacional de Educación Superior,
Ciencia, Tecnología e Innovación (SENESCYT) in
2016.
8. FUNDING
In silico identification of auxiliary genes
required for -lactam resistanceVolumen 14 N ú mero 1 - 2 023
proteins required for cell wall maintenance (36).
Notably, one of the aux genes – aux11 has high
homology with the gene lysA, which encodes
diaminopimelate decarboxylase. This protein
is required for the synthesis of the amino acid
L-lysine (32), which is part of the tetrapeptide
side chain of the PG (37). In consequence, it is
possible to speculate that, in the presence of
β-lactam antibiotics, enhanced expression of
diaminopimelate decarboxylase is crucial to
the cross-linking of PG, performed by penicillin
binding proteins.
When resistant bacteria are exposed to antibiotics,
before they synthesise antibiotic resistance
proteins such as PBP2a, they temporarily become
more vulnerable to osmotic stress. Therefore, the
bacterial cell must activate genes required for
the transport or synthesis of osmoprotectants
or compatible solutes, which contribute to
withstanding osmotic stress (38). Some putative
genes that might play that important role have
been identified in this study: cutD, gbsA and
betA. All these genes were initially identified as
aux16. The putative gene cutD was predicted to
have transmembrane domains, which correlates
with the predicted function of its product. The
gene cutD may be required for the transport of
osmoprectants such as betaine or precursors
of other osmoprotectants such as choline (39).
The putative genes gbsA and betA encode the
proteins: betaine aldehyde dehydrogenase
and choline dehydrogenase, respectively.
Betaine aldehyde dehydrogenase mediates the
biosynthesis of the osmoprotectant betaine,
whereas choline dehydrogenase is responsible
for the synthesis of the osmoprotectant glycine
betaine (40).
β-lactam antibiotics induce the expression of
operons that contain β-lactam resistance genes.
Furthermore, there is evidence that β-lactam
antibiotics also trigger a global stress response in
bacterial cells. This occurs when the cell detects
cell-wall synthesis perturbations with the aid of
the specific VraS/R two-component system (27).
In this study we identified two putative genes:
vraD and vraE (initially identified as aux19), whose
products might be part of VraS/R two-component
system. The activation of this two-component
system leads to massive overexpression of genes,
many of which are crucial for β-lactam antibiotic
resistance (27). For instance, VraS/R induces the
expression of RelP and RelQ, which synthesises
alarmones during the exponential phase of
growth (41). Alarmones are generally produced
during nutrient starvation (for instance, amino
acid starvation) and play a principal role in
stringent response, whereby the synthesis of
rRNA, tRNA and amino acids available in the
cell is repressed and synthesis of amino acids
present in insufficient amounts within the cell
is induced (42). The principal known alarmones
are guanosine tetraphosphate (ppGpp) and
guanosine pentaphosphate (pppGpp). During
nutrient starvation, these alarmones are
produced by specific proteins such as RelA or
SpoT, which become activated when uncharged
tRNA molecules bind the ribosomal site A (43).
There is evidence that this alarmones increase
the resistance of MRSA to antibiotics (44).
The global stress response requires the
participation of many different genes involved
in cascade reactions such as protein kinases,
phosphatases or transcriptional regulators.
These molecules play similar roles in prokaryotes
and eukaryotes (45). We identified three putative
genes whose products may participate in this
cascade: prpC, pknB and ccpA (initially identified
as aux1, aux2 and aux4, respectively). We can
presume that the products of these genes
may also be involved in the cascade reactions
triggered by the VraS/R two-component system,
but this proposed linkage must be verified
experimentally.
5. CONCLUSIONS
In conclusion, we can confirm that the gene
blrA is required for β-lactam resistance in the
CA-MRSA strain MW2, but does not affect the
expression of the gene mecA or the structure of
the wall teichoic acids. At the same time, many
auxiliary genes present in CA-MRSA strain MW2
may be involved in global cellular regulation,
stress response and metabolic pathways.
The author declares no conflict of interest
The author takes complete responsibility for the
information presented in this original scientific
article
6. CONFLICT OF INTEREST
7. LIMITATIONS OF LIABILITY
The present Research Project was funded by
Secretaría Nacional de Educación Superior,
Ciencia, Tecnología e Innovación (SENESCYT) in
2016.
8. FUNDING
64Igor Eduardo Astudillo Skliarova
http://revistas.espoch.edu.ec/index.php/cssn
7. Typas A, Banzhaf M, Gross CA, Vollmer
W. From the regulation of peptidoglycan
synthesis to bacterial growth and
morphology. Nature Reviews Microbiology.
2012;10(2):123-36.
8. Oliveira IA, Allonso D, Fernandes TVA,
Lucena DMS, Ventura GT, Dias WB, et al.
Enzymatic and structural properties of
human glutamine:fructose-6-phosphate
amidotransferase 2 (hGFAT2). J Biol Chem.
2021;296:100180.
9. Hummels KR, Berry SP, Li Z, Taguchi A, Min
JK, Walker S, et al. Coordination of bacterial
cell wall and outer membrane biosynthesis.
Nature. 2023;615(7951):300-4.
10. Hrast M, Rožman K, Ogris I, Škedelj V, Patin
D, Sova M, et al. Evaluation of the published
kinase inhibitor set to identify multiple
inhibitors of bacterial ATP-dependent
mur ligases. J Enzyme Inhib Med Chem.
2019;34(1):1010-7.
11. Song Y, Lee JS, Shin J, Lee GM, Jin S,
Kang S, et al. Functional cooperation
of the glycine synthase-reductase and
Wood–Ljungdahl pathways for
autotrophic growth of <i>Clostridium
drakei</i>. Proceedings of the National
Academy of Sciences. 2020;117(13):7516-
23.
12. Monteiro JM, Münch D, Filipe SR, Schneider
T, Sahl H-G, Pinho MG. The pentaglycine
bridges of <em>Staphylococcus aureus</
em> peptidoglycan are essential for cell
integrity. bioRxiv. 2018:479006.
13. Travis BA, Peck JV, Salinas R, Dopkins
B, Lent N, Nguyen VD, et al. Molecular
dissection of the glutamine synthetase-
GlnR nitrogen regulatory circuitry in Gram-
positive bacteria. Nature Communications.
2022;13(1):3793.
14. Nöldeke ER, Muckenfuss LM, Niemann V,
Müller A, Störk E, Zocher G, et al. Structural
basis of cell wall peptidoglycan amidation by
the GatD/MurT complex of Staphylococcus
aureus. Sci Rep. 2018;8(1):12953.
15. Kuk ACY, Hao A, Lee S-Y. Structure
and Mechanism of the Lipid Flippase
MurJ. Annual Review of Biochemistry.
2022;91(1):705-29.
16. Liu X, Meiresonne NY, Bouhss A, den
Blaauwen T. FtsW activity and lipid II
1. Kwiecinski JM, Horswill AR. Staphylococcus
aureus bloodstream infections: pathogenesis
and regulatory mechanisms. Curr Opin
Microbiol. 2020;53:51-60.
2. Oliveira K, Viegas C, Ribeiro E. MRSA
Colonization in Workers from Different
Occupational Environments—A One
Health Approach Perspective. Atmosphere.
2022;13(5):658.
3. Dittmann K, Schmidt T, Müller G, Cuny C,
Holtfreter S, Troitzsch D, et al. Susceptibility
of livestock-associated methicillin-resistant
Staphylococcus aureus (LA-MRSA) to
chlorhexidine digluconate, octenidine
dihydrochloride, polyhexanide, PVP-iodine
and triclosan in comparison to hospital-
acquired MRSA (HA-MRSA) and community-
aquired MRSA (CA-MRSA): a standardized
comparison. Antimicrob Resist Infect
Control. 2019;8:122.
4. Turner NA, Sharma-Kuinkel BK, Maskarinec
SA, Eichenberger EM, Shah PP, Carugati M,
et al. Methicillin-resistant Staphylococcus
aureus: an overview of basic and clinical
research. Nature Reviews Microbiology.
2019;17(4):203-18.
5. Okiki PA, Eromosele ES, Ade-Ojo P, Sobajo
OA, Idris OO, Agbana RD. Occurrence of
mecA and blaZ genes in methicillin-resistant
Staphylococcus aureus associated with
vaginitis among pregnant women in Ado-
Ekiti, Nigeria. New Microbes New Infect.
2020;38:100772.
6. Mikkelsen K, Sirisarn W, Alharbi O, Alharbi
M, Liu H, Nøhr-Meldgaard K, et al. The
Novel Membrane-Associated Auxiliary
Factors AuxA and AuxB Modulate
β-lactam Resistance in MRSA by stabilizing
Lipoteichoic Acids. International Journal of
Antimicrobial Agents. 2021;57(3):106283.
6. REFERENCES
I would like to thank my former MSc supervisor
Dr. Guoqing Xia for providing feedback on the
text, figures and tables of this original scientific
article. Additionally, I would like to express my
gratitude to the Faculty of Public Health (Facultad
de Salud Pública) for allowing me to share the
knowledge about putative auxiliary factors
present in Staphylococcus aureus.
9. ACKNOWLEDGMENT
http://revistas.espoch.edu.ec/index.php/cssn
7. Typas A, Banzhaf M, Gross CA, Vollmer
W. From the regulation of peptidoglycan
synthesis to bacterial growth and
morphology. Nature Reviews Microbiology.
2012;10(2):123-36.
8. Oliveira IA, Allonso D, Fernandes TVA,
Lucena DMS, Ventura GT, Dias WB, et al.
Enzymatic and structural properties of
human glutamine:fructose-6-phosphate
amidotransferase 2 (hGFAT2). J Biol Chem.
2021;296:100180.
9. Hummels KR, Berry SP, Li Z, Taguchi A, Min
JK, Walker S, et al. Coordination of bacterial
cell wall and outer membrane biosynthesis.
Nature. 2023;615(7951):300-4.
10. Hrast M, Rožman K, Ogris I, Škedelj V, Patin
D, Sova M, et al. Evaluation of the published
kinase inhibitor set to identify multiple
inhibitors of bacterial ATP-dependent
mur ligases. J Enzyme Inhib Med Chem.
2019;34(1):1010-7.
11. Song Y, Lee JS, Shin J, Lee GM, Jin S,
Kang S, et al. Functional cooperation
of the glycine synthase-reductase and
Wood–Ljungdahl pathways for
autotrophic growth of <i>Clostridium
drakei</i>. Proceedings of the National
Academy of Sciences. 2020;117(13):7516-
23.
12. Monteiro JM, Münch D, Filipe SR, Schneider
T, Sahl H-G, Pinho MG. The pentaglycine
bridges of <em>Staphylococcus aureus</
em> peptidoglycan are essential for cell
integrity. bioRxiv. 2018:479006.
13. Travis BA, Peck JV, Salinas R, Dopkins
B, Lent N, Nguyen VD, et al. Molecular
dissection of the glutamine synthetase-
GlnR nitrogen regulatory circuitry in Gram-
positive bacteria. Nature Communications.
2022;13(1):3793.
14. Nöldeke ER, Muckenfuss LM, Niemann V,
Müller A, Störk E, Zocher G, et al. Structural
basis of cell wall peptidoglycan amidation by
the GatD/MurT complex of Staphylococcus
aureus. Sci Rep. 2018;8(1):12953.
15. Kuk ACY, Hao A, Lee S-Y. Structure
and Mechanism of the Lipid Flippase
MurJ. Annual Review of Biochemistry.
2022;91(1):705-29.
16. Liu X, Meiresonne NY, Bouhss A, den
Blaauwen T. FtsW activity and lipid II
1. Kwiecinski JM, Horswill AR. Staphylococcus
aureus bloodstream infections: pathogenesis
and regulatory mechanisms. Curr Opin
Microbiol. 2020;53:51-60.
2. Oliveira K, Viegas C, Ribeiro E. MRSA
Colonization in Workers from Different
Occupational Environments—A One
Health Approach Perspective. Atmosphere.
2022;13(5):658.
3. Dittmann K, Schmidt T, Müller G, Cuny C,
Holtfreter S, Troitzsch D, et al. Susceptibility
of livestock-associated methicillin-resistant
Staphylococcus aureus (LA-MRSA) to
chlorhexidine digluconate, octenidine
dihydrochloride, polyhexanide, PVP-iodine
and triclosan in comparison to hospital-
acquired MRSA (HA-MRSA) and community-
aquired MRSA (CA-MRSA): a standardized
comparison. Antimicrob Resist Infect
Control. 2019;8:122.
4. Turner NA, Sharma-Kuinkel BK, Maskarinec
SA, Eichenberger EM, Shah PP, Carugati M,
et al. Methicillin-resistant Staphylococcus
aureus: an overview of basic and clinical
research. Nature Reviews Microbiology.
2019;17(4):203-18.
5. Okiki PA, Eromosele ES, Ade-Ojo P, Sobajo
OA, Idris OO, Agbana RD. Occurrence of
mecA and blaZ genes in methicillin-resistant
Staphylococcus aureus associated with
vaginitis among pregnant women in Ado-
Ekiti, Nigeria. New Microbes New Infect.
2020;38:100772.
6. Mikkelsen K, Sirisarn W, Alharbi O, Alharbi
M, Liu H, Nøhr-Meldgaard K, et al. The
Novel Membrane-Associated Auxiliary
Factors AuxA and AuxB Modulate
β-lactam Resistance in MRSA by stabilizing
Lipoteichoic Acids. International Journal of
Antimicrobial Agents. 2021;57(3):106283.
6. REFERENCES
I would like to thank my former MSc supervisor
Dr. Guoqing Xia for providing feedback on the
text, figures and tables of this original scientific
article. Additionally, I would like to express my
gratitude to the Faculty of Public Health (Facultad
de Salud Pública) for allowing me to share the
knowledge about putative auxiliary factors
present in Staphylococcus aureus.
9. ACKNOWLEDGMENT
65
In silico identification of auxiliary genes
required for -lactam resistanceVolumen 14 N ú mero 1 - 2 023
synthesis are required for recruitment
of MurJ to midcell during cell division in
Escherichia coli. Molecular Microbiology.
2018;109(6):855-84.
17. Wacnik K, Rao VA, Chen X, Lafage L, Pazos
M, Booth S, et al. Penicillin-Binding Protein
1 (PBP1) of Staphylococcus aureus Has
Multiple Essential Functions in Cell Division.
mBio. 2022;13(4):e0066922.
18. da Costa TM, de Oliveira CR, Chambers HF,
Chatterjee SS. PBP4: A New Perspective on
Staphylococcus aureus β-Lactam Resistance.
Microorganisms. 2018;6(3).
19. Roch M, Lelong E, Panasenko OO, Sierra
R, Renzoni A, Kelley WL. Thermosensitive
PBP2a requires extracellular folding factors
PrsA and HtrA1 for Staphylococcus aureus
MRSA β-lactam resistance. Commun Biol.
2019;2:417.
20. Dalal V, Kumar P, Rakhaminov G, Qamar
A, Fan X, Hunter H, et al. Repurposing an
Ancient Protein Core Structure: Structural
Studies on FmtA, a Novel Esterase of
Staphylococcus aureus. Journal of Molecular
Biology. 2019;431(17):3107-23.
21. G CB, Sahukhal GS, Elasri MO. Role of the
msaABCR Operon in Cell Wall Biosynthesis,
Autolysis, Integrity, and Antibiotic Resistance
in Staphylococcus aureus. Antimicrob
Agents Chemother. 2019;63(10).
22. Walter A, Unsleber S, Rismondo J, Jorge AM,
Peschel A, Gründling A, et al. Phosphoglycerol-
type wall and lipoteichoic acids are
enantiomeric polymers differentiated by the
stereospecific glycerophosphodiesterase
GlpQ. J Biol Chem. 2020;295(12):4024-34.
23. Chee Wezen X, Chandran A, Eapen RS,
Waters E, Bricio-Moreno L, Tosi T, et al.
Structure-Based Discovery of Lipoteichoic
Acid Synthase Inhibitors. Journal of
Chemical Information and Modeling.
2022;62(10):2586-99.
24. Vickery CR, Wood BM, Morris HG, Losick R,
Walker S. Reconstitution of Staphylococcus
aureus Lipoteichoic Acid Synthase Activity
Identifies Congo Red as a Selective Inhibitor.
Journal of the American Chemical Society.
2018;140(3):876-9.
25. Zeng J, Platig J, Cheng TY, Ahmed S, Skaf Y,
Potluri LP, et al. Protein kinases PknA and PknB
independently and coordinately regulate
essential Mycobacterium tuberculosis
physiologies and antimicrobial susceptibility.
PLoS Pathog. 2020;16(4):e1008452.
26. Jenul C, Horswill AR. Regulation of
Staphylococcus aureus Virulence. Microbiol
Spectr. 2019;7(2).
27. Bleul L, Francois P, Wolz C. Two-Component
Systems of S. aureus: Signaling and Sensing
Mechanisms. Genes (Basel). 2021;13(1).
28. Miragaia M. Factors Contributing to the
Evolution of mecA-Mediated β-lactam
Resistance in Staphylococci: Update and New
Insights From Whole Genome Sequencing
(WGS). Frontiers in Microbiology. 2018;9.
29. Tooke CL, Hinchliffe P, Bragginton EC,
Colenso CK, Hirvonen VHA, Takebayashi
Y, et al. β-Lactamases and β-Lactamase
Inhibitors in the 21st Century. J Mol Biol.
2019;431(18):3472-500.
30. Yadav AK, Espaillat A, Cava F. Bacterial
Strategies to Preserve Cell Wall Integrity
Against Environmental Threats. Front
Microbiol. 2018;9:2064.
31. De Lencastre H, Wu SW, Pinho MG,
Ludovice AM, Filipe S, Gardete S, et al.
Antibiotic resistance as a stress response:
complete sequencing of a large number of
chromosomal loci in Staphylococcus aureus
strain COL that impact on the expression of
resistance to methicillin. Microb Drug Resist.
1999;5(3):163-75.
32. Xu J-Z, Ruan H-Z, Liu L-M, Wang L-P, Zhang
W-G. Overexpression of thermostable
meso-diaminopimelate dehydrogenase
to redirect diaminopimelate pathway for
increasing L-lysine production in Escherichia
coli. Scientific Reports. 2019;9(1):2423.
33. Boyd SE, Livermore DM, Hooper DC, Hope
WW. Metallo-β-Lactamases: Structure,
Function, Epidemiology, Treatment
Options, and the Development Pipeline.
Antimicrobial Agents and Chemotherapy.
2020;64(10):e00397-20.
34. Aslanli A, Domnin M, Stepanov N, Efremenko
E. "Universal" Antimicrobial Combination of
Bacitracin and His(6)-OPH with Lactonase
Activity, Acting against Various Bacterial and
Yeast Cells. Int J Mol Sci. 2022;23(16).
35. Berger-Bächi B. Insertional inactivation of
staphylococcal methicillin resistance by
In silico identification of auxiliary genes
required for -lactam resistanceVolumen 14 N ú mero 1 - 2 023
synthesis are required for recruitment
of MurJ to midcell during cell division in
Escherichia coli. Molecular Microbiology.
2018;109(6):855-84.
17. Wacnik K, Rao VA, Chen X, Lafage L, Pazos
M, Booth S, et al. Penicillin-Binding Protein
1 (PBP1) of Staphylococcus aureus Has
Multiple Essential Functions in Cell Division.
mBio. 2022;13(4):e0066922.
18. da Costa TM, de Oliveira CR, Chambers HF,
Chatterjee SS. PBP4: A New Perspective on
Staphylococcus aureus β-Lactam Resistance.
Microorganisms. 2018;6(3).
19. Roch M, Lelong E, Panasenko OO, Sierra
R, Renzoni A, Kelley WL. Thermosensitive
PBP2a requires extracellular folding factors
PrsA and HtrA1 for Staphylococcus aureus
MRSA β-lactam resistance. Commun Biol.
2019;2:417.
20. Dalal V, Kumar P, Rakhaminov G, Qamar
A, Fan X, Hunter H, et al. Repurposing an
Ancient Protein Core Structure: Structural
Studies on FmtA, a Novel Esterase of
Staphylococcus aureus. Journal of Molecular
Biology. 2019;431(17):3107-23.
21. G CB, Sahukhal GS, Elasri MO. Role of the
msaABCR Operon in Cell Wall Biosynthesis,
Autolysis, Integrity, and Antibiotic Resistance
in Staphylococcus aureus. Antimicrob
Agents Chemother. 2019;63(10).
22. Walter A, Unsleber S, Rismondo J, Jorge AM,
Peschel A, Gründling A, et al. Phosphoglycerol-
type wall and lipoteichoic acids are
enantiomeric polymers differentiated by the
stereospecific glycerophosphodiesterase
GlpQ. J Biol Chem. 2020;295(12):4024-34.
23. Chee Wezen X, Chandran A, Eapen RS,
Waters E, Bricio-Moreno L, Tosi T, et al.
Structure-Based Discovery of Lipoteichoic
Acid Synthase Inhibitors. Journal of
Chemical Information and Modeling.
2022;62(10):2586-99.
24. Vickery CR, Wood BM, Morris HG, Losick R,
Walker S. Reconstitution of Staphylococcus
aureus Lipoteichoic Acid Synthase Activity
Identifies Congo Red as a Selective Inhibitor.
Journal of the American Chemical Society.
2018;140(3):876-9.
25. Zeng J, Platig J, Cheng TY, Ahmed S, Skaf Y,
Potluri LP, et al. Protein kinases PknA and PknB
independently and coordinately regulate
essential Mycobacterium tuberculosis
physiologies and antimicrobial susceptibility.
PLoS Pathog. 2020;16(4):e1008452.
26. Jenul C, Horswill AR. Regulation of
Staphylococcus aureus Virulence. Microbiol
Spectr. 2019;7(2).
27. Bleul L, Francois P, Wolz C. Two-Component
Systems of S. aureus: Signaling and Sensing
Mechanisms. Genes (Basel). 2021;13(1).
28. Miragaia M. Factors Contributing to the
Evolution of mecA-Mediated β-lactam
Resistance in Staphylococci: Update and New
Insights From Whole Genome Sequencing
(WGS). Frontiers in Microbiology. 2018;9.
29. Tooke CL, Hinchliffe P, Bragginton EC,
Colenso CK, Hirvonen VHA, Takebayashi
Y, et al. β-Lactamases and β-Lactamase
Inhibitors in the 21st Century. J Mol Biol.
2019;431(18):3472-500.
30. Yadav AK, Espaillat A, Cava F. Bacterial
Strategies to Preserve Cell Wall Integrity
Against Environmental Threats. Front
Microbiol. 2018;9:2064.
31. De Lencastre H, Wu SW, Pinho MG,
Ludovice AM, Filipe S, Gardete S, et al.
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Modulation of Peptidoglycan Synthesis by
Recycled Cell Wall Tetrapeptides. Cell Rep.
2020;31(4):107578.
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S, Pottier S, Dolivet A, et al. Intracellular
osmoprotectant concentrations determine
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during drying. Applied Microbiology and
Biotechnology. 2020;104(7):3145-56.
39. Yang N, Ding R, Liu J. Synthesizing glycine
betaine via choline oxidation pathway as an
osmoprotectant strategy in Haloferacales.
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and Related Osmoprotectants. Targets for
Metabolic Engineering of Stress Resistance1.
Plant Physiology. 1999;120(4):945-9.
41. Salzer A, Keinhörster D, Kästle C, Kästle B,
Wolz C. Small Alarmone Synthetases RelP and
RelQ of Staphylococcus aureus Are Involved
in Biofilm Formation and Maintenance
Under Cell Wall Stress Conditions. Front
Microbiol. 2020;11:575882.
42. Magnusson LU, Farewell A, Nyström T.
ppGpp: a global regulator in Escherichia coli.
Trends Microbiol. 2005;13(5):236-42.
43. Kudrin P, Dzhygyr I, Ishiguro K, Beljantseva
J, Maksimova E, Oliveira SRA, et al. The
ribosomal A-site finger is crucial for binding
and activation of the stringent factor RelA.
Nucleic Acids Res. 2018;46(4):1973-83.
44. Fritsch VN, Loi VV, Busche T, Tung QN,
Lill R, Horvatek P, et al. The alarmone (p)
ppGpp confers tolerance to oxidative
stress during the stationary phase by
maintenance of redox and iron homeostasis
in Staphylococcus aureus. Free Radic Biol
Med. 2020;161:351-64.
45. Madsen CD, Hein J, Workman CT. Systematic
inference of indirect transcriptional
regulation by protein kinases and
phosphatases. PLoS Comput Biol.
2022;18(6):e1009414.