BUSCADOR RECOLECTA

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI) characterized
by bacterial biofilm formation and recalcitrance to immune-mediated clearance and antibiotics.
The molecular events behind PJI infection are yet to be unraveled. In this sense, identification of
polymorphisms in bacterial genomes may help to establish associations between sequence variants
and the ability of S. aureus to cause PJI. Here, we report an experimental nucleotide-level survey
specifically aimed at the intergenic regions (IGRs) of the icaADBCR locus, which is responsible for
the synthesis of the biofilm exopolysaccharide PIA/PNAG, in a collection of strains sampled from
PJI and wounds. IGRs of the icaADBCR locus were highly conserved and no PJI-specific SNPs were
found. Moreover, polymorphisms in these IGRs did not significantly affect transcription of the
icaADBC operon under in vitro laboratory conditions. In contrast, an SNP within the icaR coding
region, resulting in a V176E change in the transcriptional repressor IcaR, led to a significant increase
in icaADBC operon transcription and PIA/PNAG production and a reduction in S. aureus virulence
in a Galleria mellonella infection model. In conclusion, SNPs in icaADBCR IGRs of S. aureus isolates
from PJI are not associated with icaADBC expression, PIA/PNAG production and adaptation to PJI., This work was financially supported by the Spanish Ministry of Science and Innovation
grant PID2020-113494RB-I00 to I.L. (Agencia Española de Investigación/Fondo Europeo de Desarrollo
Regional, European Union), the European Union’s Horizon 2020 research and innovation program
under the Marie Skłodowska-Curie grant agreement No 754412 [MoRE2020—Region Västra Götaland],
and CARe—Centre for Antibiotic Resistance Research at University of Gothenburg. L.M.-L. was supported by the European Union’s H2020 research and innovation programme under Marie
Sklodowska-Curie grant agreement No 801586 (IberusTalent).

In this thesis, we have focused on studying variants found in IGRs adjacent to the most important genes involved in S. aureus biofilm formation; the icaADBCR locus, and the genes encoding the family of surface adhesins. For this purpose, we sequenced the whole genome of a collection of 71 S. aureus isolates from periprosthetic joint infections (PJI) and wound infections stored at the Clinical Bacteriological Laboratory of the Sahlgrenska University Hospital and at the Culture Collection University of Gothenburg (CCUG), respectively.
In the first chapter, we explored the regulatory regions of the icaADBCR locus to identify patterns that might be associated with an increased capacity of the isolates to produce PIA/PNAG and form a biofilm. This study compared the regulatory regions of the icaADBCR locus in the genomes of PJI and wound isolates with those in the genome of the reference strain MW2. From these analyses, strains were grouped based on the SNPs found in the IGRs of the operon and also within the coding region of the transcriptional regulator IcaR.
These regions showed high conservation rates, and no pattern associated with the origin of the isolates, either PJI or wounds, was detected. On the other hand, using transcriptional fusions between the regulatory region of the icaADBCR locus and the green fluorescent protein gene (gfp), we demonstrated that the expression of icaADBC genes was not affected by the presence of variations in IGRs. Notably, a SNP within the coding region of icaR, which results in an amino acid change in the transcriptional repressor IcaR V176E, led to a significant increase in the transcription of the icaADBC operon and the production of PIA/PNAG. Using a Galleria mellonella infection model, we were able to demonstrate a significant reduction in S. aureus virulence associated with the
increase in PIA/PNAG production.
In the second chapter, we focused on analyzing the association between SNPs in the promoter regions of genes encoding adhesion-related proteins with adhesins expression levels and therefore, the ability of the strain to adhere to medical devices. Genome analyses of PJI and wound isolates showed different profiles in the content of adhesin-encoding genes. Some of these, such as sasG and cna, were lineage-associated, and fifteen genes were present in the whole collection of strains. When the variability in the SNPs contained in regulatory regions that control the expression of each adhesin was investigated, different variation rates were found among the isolates. Following the same approach as in chapter I, based on transcriptional fusions between regulatory regions and the gfp gene, results showed that each genetic lineage contained a specific profile of adhesins expression under the same environmental condition.
Moreover, we developed a biomaterial-associated murine infection model together with a metagenomic analysis to simultaneously compare the capacity of different S. aureus isolates to colonize medical implants.
In summary, our results evidenced that SNPs in the IGRs flanking the genes encoding factors important for biofilm development may contribute to the generation of variability in the capacity of S. aureus to colonize medical implants.
In particular, our results revealed that IGRs controlling the expression of the icaADBC locus and production of the PIA/PNAG exopolysaccharide are highly conserved and that very few silent SNPs can be detected between strains. On the
contrary, SNPs in the IGRs of genes encoding surface adhesins provide a profile of proteins expression that is specific for each S. aureus clonal complex (CC).
Altogether, these studies emphasize the importance of investigating the potential impact of SNPs inside IGRs on gene expression and specific bacterial traits, such as pathogen colonization success., European Union's H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 801586; Spanish Ministry of Economy, Industry and Competitiveness grant BIO2017-83035-R; Spanish Ministry of Science and Innovation grant PID2020-113494RB-I00., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Staphylococcus aureus es una bacteria versátil que se puede encontrar estableciendo una relación comensal en la piel de, aproximadamente, el 30% de la población sin causar ningún problema. Sin embargo, cuando ésta atraviesa la barrera epitelial y dependiendo del estado inmunológico del portador, puede causar patologías leves, como los abscesos, o severas, como la endocarditis u osteomielitis. Para el tratamiento de estas patologías, existe un amplio abanico de fármacos, no obstante, S. aureus ha desarrollado mecanismos que le permiten incrementar su resistencia y evadir el efecto de estos antibióticos.
En el capítulo I de esta tesis doctoral, hemos analizado la implicación de los TCSs de S. aureus en el posible desarrollo de resistencia a dos antibióticos glicopéptidos de reciente descubrimiento, la complestatina (Cm) y la corbomicina (Cb). Para ello, hemos estudiado la susceptibilidad frente a ambos antibióticos de una colección compuesta por mutantes simples en cada TCS en la cepa MW2 y otra compuesta por derivados de una cepa mutante múltiple en todos los TCSs no esenciales (ΔXV) complementados con un plásmido portador de un único TCS. Con ayuda de estas colecciones, observamos que el sistema VraSR es el único que controla la susceptibilidad de S. aureus a la Cm y Cb sugiriendo que uno o varios componentes específicos del regulón de VraSR podrían ser los responsables de este control. Estudiando el regulón de VraSR en profundidad, determinamos que la regulación directa de SpdC e indirecta de SagB por parte de este TCS podrían estar relacionadas con la menor susceptibilidad que presentan las cepas portadoras del sistema VraSR. Al estar SpdC y SagB implicados en la longitud de las cadenas de azúcar del peptidoglicano, analizamos la posible presencia de cambios en la composición del mismo en los mutantes objeto de estudio. De entre las cepas estudiadas, se apreció un incremento en la cantidad de muropéptido compuesto por dímeros M5-5Gly-M4-1Ala en la cepa mutante en vraSR, pero no se pudo demostrar si este cambio está relacionado con la mayor susceptibilidad de esta cepa a la Cm y Cb.
Por otra parte, el capítulo II está dedicado a revisar las evidencias que demuestran la regulación ejercida por los TCSs en su estado no fosforilado, lo que supone un cambio de paradigma en la regulación de los TCSs.
Finalmente, en el capítulo III, hemos analizado la regulación de la expresión del operón icaADBC y de otros factores de virulencia por parte del TCS ArlRS. En concreto, analizamos como la forma fosforilada y no fosforilada de ArlR regulan la expresión de icaADBC y del represor icaR, responsables de la producción del exopolisacárido PIA/PNAG, elemento mayoritario del biofilm de S. aureus. Los resultados indicaron que la forma no fosforilada de ArlR actúa como represor de icaR causando un aumento de la producción de las proteínas IcaADBC y un aumento de la producción de PIA/PNAG., Staphylococcus aureus is a versatile bacterium that can be found establishing a commensal relationship on the skin of approximately 30% of the population without causing any harm. However, when it crosses the epithelial barrier and depending on the immune status of the carrier, it can cause mild pathologies such as abscesses, or severe ones, such as endocarditis or osteomyelitis. For the treatment of these pathologies, there is a wide range of available drugs. Nevertheless, S. aureus has developed mechanisms that allow it to increase its resistance and evade the effect of these antibiotics.
In chapter I, we have analyzed the implication of the TCSs of S. aureus in the possible development of resistance against two recently discovered glycopeptide antibiotics, complestatin (Cm) and corbomycin (Cb). For this purpose, we tested the susceptibility to both antibiotics of a collection of single mutants in each TCS in the MW2 strain and another collection composed of derivatives of a multiple mutant in each non essential TCS (ΔXV strain) complemented with a plasmid carrying a single TCS. Using these collections, we observed that the VraSR system is the only TCS that controls the susceptibility of S. aureus to Cm and Cb, suggesting that one or several specific components of the VraSR regulon might be responsible for this control. By futher studying the VraSR regulon, we determined that the direct regulation of SpdC and the indirect regulation of SagB by this TCS might be related to the lower susceptibility phenotype shown by the strains carrying the VraSR system. Since SpdC and SagB are involved in the length of the peptidoglycan chains, we analysed the presence of changes in the peptidoglycan in the mutants under study. Among the strains analysed, the vraSR mutant showed an increase in the amount of muropeptide composed of M5-5Gly-M4-1Ala dimers, however, it could not be demonstrated whether this change is related to the increased susceptibility to Cm and Cb.
On the other hand, chapter II is aimed at reviewing the literature related to the regulation exerted by TCSs in their non-phosphorylated state, which represents a paradigm shift in TCS regulation.
Finally, in chapter III, we have analysed the regulation of the expression of the icaADBC operon and other virulence factors. Specifically, we analysed how the phosphorylated and non-phosphorylated forms of ArlR regulate the expression of the icaR repressor and icaADBC operon, responsible for the production of the exopolysaccharide PIA/PNAG, the main component of the S. aureus biofilm. Results indicated that the non-phosphorylated form of ArlR acts as a repressor of icaR causing an increased production of IcaADBC and PIA/PNAG., Este trabajo ha sido realizado dentro de los proyectos de investigación:
• Título del proyecto: ‘Caracterización funcional de los determinantes moleculares para la adaptación de Staphylococcus aureus a la virulencia’. BIO2017-83035-R. Ministerio de Economía, Industria y Competitividad.
• Título del proyecto: ‘Operones no-contiguos: un nuevo nivel de regulación génica en bacterias’. PID2020-113494RB-100. Ministerio de Ciencia e Innovación., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Sonicating explanted prosthetic implants to physically remove biofilms is a recognized method for improving the microbiological diagnosis of prosthetic joint infection (PJI); however, chemical and enzymatic treatments have been investigated as alternative biofilm removal methods. We compared the biofilm dislodging efficacy of sonication followed by the addition of enzyme cocktails with different activity spectra in the diagnosis of PJI with that of the sonication of fluid cultures alone. Consecutive patients who underwent prosthesis explantation due to infection at our institution were prospectively enrolled for 1 year. The diagnostic procedure included the collection of five intraoperative tissue cultures, sonication of the removed devices, and conventional culture of the sonication fluid. The resulting sonication fluid was also treated with an enzyme cocktail consisting of homemade dispersin B (0.04 ¿g/mL) and proteinase K (Sigma; 100 ¿g/mL) for 45 minutes at 37°C. The resulting sonication (S) and sonication with subsequent enzymatic treatment (SE) fluids were plated for aerobic and anaerobic culture broth for 7 days (aerobic) or 14 days (anaerobic). Identification was performed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Bruker). We included 107 patients from whom a prosthetic implant had been removed, among which PJI was diagnosed in 36 (34%). The sensitivity of S alone was significantly greater than that of SE alone (82% vs 71%; P < 0.05). Four patients with PJI were positive after sonication alone but negative after sonication plus enzymatic treatment. The four microorganisms missed after the addition of the enzyme cocktail were Staphylococcus aureus, two coagulase-negative Staphylococci, and Cutibacterium acnes. In conclusion, sonication alone was more sensitive than sonication followed by enzymatic treatment. The combination of these two methods had no synergistic effect; in contrast, the results suggest that the combination of both dislodgment methods affects the viability of gram-positive microorganisms., This study was supported by a project funded by the Government of Navarra. Proyecto DIPAN Gº Na 56/22. Principal investigator: M.E. Portillo. And by the Spanish Ministry of Science, Innovation and Universities grant PID2020-113494RB-I00 AEI/10.13039/501100011033 (Agencia Española de Investigación Fondo Europeo de Desarrollo Regional, European Union) to I.L.

Dormant prophages protect lysogenic cells by expressing diverse immune systems, which must avoid targeting their cognate prophages upon activation. Here we report that multiple Staphylococcus aureus prophages encode Tha (tail-activated, HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain-containing anti-phage system), a defence system activated by structural tail proteins of incoming phages. We demonstrate the function of two Tha systems, Tha-1 and Tha-2, activated by distinct tail proteins. Interestingly, Tha systems can also block reproduction of the induced tha-positive prophages. To prevent autoimmunity after prophage induction, these systems are inhibited by the product of a small overlapping antisense gene previously believed to encode an excisionase. This genetic organization, conserved in S. aureus prophages, allows Tha systems to protect prophages and their bacterial hosts against phage predation and to be turned of during prophage induction, balancing immunity and autoimmunity. Our results show that the fne regulation of these processes is essential for the correct development of prophages’ life cycle., This work was supported by grants MR/X020223/1, MR/M003876/1, MR/V000772/1 and MR/S00940X/1 from the Medical Research Council (UK); BB/V002376/1 and BB/V009583/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK); and EP/X026671/1 from the Engineering and Physical Sciences Research Council (EPSRC, UK) and grant PID2020-113494RB-I00 from the Spanish Ministry of Science and Innovation (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union).

Biodegradable polymeric coatings are being explored as a preventive strategy for orthopaedic device-related infection. In this study, titanium surfaces (Ti) were coated with poly-D,L-lactide (PDLLA, (P)), polyethylene-glycol poly-D,L-lactide (PEGylated-PDLLA, (PP20)), or multi-layered PEGylated-PDLLA (M), with or without 1 % silver sulfadiazine. The aim was to evaluate their cytocompatibility, resistance to Staphylococcus aureus biofilm formation, and their potential to enhance the susceptibility of any biofilm formed to antibiotics. Using automated high-content screening confocal microscopy, biofilm formation of a clinical methicillin-resistant Staphylococcus aureus (MRSA) isolate expressing GFP was quantified, along with isogenic mutants that were unable to form polysaccharidic or proteinaceous biofilm matrices. The results showed that PEGylated-PDLLA coatings exhibited significant antibiofilm properties, with M showing the highest effect. This inhibitory effect was stronger in S. aureus biofilms with a matrix composed of proteins compared to those with an exopolysaccharide (PIA) biofilm matrix. Our data suggest that the antibiofilm effect may have been due to (i) inhibition of the initial attachment through microbial surface components recognising adhesive matrix molecules (MSCRAMMs), since PEG reduces protein surface adsorption via surface hydration layer and steric repulsion; and (ii) mechanical disaggregation and dispersal of microcolonies due to the bioresorbable/degradable nature of the polymers, which undergo hydration and hydrolysis over time. The disruption of biofilm morphology by the PDLLA-PEG co-polymers increased S. aureus susceptibility to antibiotics like rifampicin and fusidic acid. Adding 1 % AgSD provided additional early bactericidal effects on both biofilm and planktonic S. aureus. Additionally, the coatings were cytocompatible with immune cells, indicating their potential to enhance bacterial clearance and reduce bacterial colonisation of titanium-based orthopaedic biomaterials., This research was funded by the European Commission within the H2020- MSCA grant agreement No. 861046 (BIOREMIA-ETN); Swedish Research Council (2022-00853); the Swedish state under the agreement between the Swedish government and the county councils; the ALF agreement (ALFGBG-978896); the IngaBritt and Arne Lundberg Foundation (LU2021-0048); the Hjalmar Svensson Foundation; the Doctor Felix Neuberghs Foundation; the Adlerbertska Foundation; the Area of Advance Materials of Chalmers/ GU Biomaterials within the Strategic Research Area initiative launched by the Swedish government; the Spanish Ministry of Science, Innovation and Universities grant PID2020-113494RB-I00 (Agencia Española de Investigación / Fondo Europeo de Desarrollo Regional , European Union) to I.L.; and S.O. was funded through the Government of Ireland Postgraduate Scholarship Programme by the Irish Research Council (GOIPG/2023/3290).

The overuse of antibiotics in humans and livestock has driven the emergence and spread of antimicrobial resistance and has therefore prompted research
on the discovery of novel antibiotics. Complestatin (Cm) and corbomycin (Cb) are
glycopeptide antibiotics with an unprecedented mechanism of action that is active
even against methicillin-resistant and daptomycin-resistant Staphylococcus aureus. They
bind to peptidoglycan and block the activity of peptidoglycan hydrolases required for
remodeling the cell wall during growth. Bacterial signaling through two-component
transduction systems (TCSs) has been associated with the development of S. aureus
antimicrobial resistance. However, the role of TCSs in S. aureus susceptibility to Cm
and Cb has not been previously addressed. In this study, we determined that, among
all 16 S. aureus TCSs, VraSR is the only one controlling the susceptibility to Cm and
Cb. Deletion of vraSR increased bacterial susceptibility to both antibiotics. Epistasis
analysis with members of the vraSR regulon revealed that deletion of spdC, which
encodes a membrane protein that scaffolds SagB for cleavage of peptidoglycan strands
to achieve physiological length, in the vraSR mutant restored Cm and Cb susceptibility
to wild-type levels. Moreover, deletion of either spdC or sagB in the wild-type strain
increased resistance to both antibiotics. Further analyses revealed a significant rise in
the relative amount of peptidoglycan and its total degree of cross-linkage in ΔspdC and
ΔsagB mutants compared to the wild-type strain, suggesting that these changes in the
cell wall provide resistance to the damaging effect of Cm and Cb., This work was financially supported by the Spanish Ministry of Science and Innovation grant PID2020-113494RB-I00 to I.L. (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) and a Canadian Institutes of Health Research grant (FRN-148463; to G.D.W.).
C.G.A. was supported by a predoctoral contract from the Public University of Navarra. Research in the Cava lab was supported by The Swedish Research Council (VR), The Knut and Alice Wallenberg Foundation (KAW), The Laboratory of Molecular Infection Medicine Sweden (MIMS) and The Kempe Foundation.

Biofilm engineering has emerged as a controllable way to fabricate living structures with programmable functionalities. The amyloidogenic proteins comprising the biofilms can be engineered to create self-assembling extracellular functionalized surfaces. In this regard, facultative amyloids, which play a dual role in biofilm formation by acting as adhesins in their native conformation and as matrix scaffolds when they polymerize into amyloid-like fibrillar structures, are interesting candidates. Here, we report the use of the facultative amyloid-like Bap protein of Staphylococcus aureus as a tool to decorate the extracellular biofilm matrix or the bacterial cell surface with a battery of functional domains or proteins. We demonstrate that the localization of the functional tags can be change by simply modulating the pH of the medium. Using Bap features, we build a tool for trapping and covalent immobilizing molecules at bacterial cell surface or at the biofilm matrix based on the SpyTag/SpyCatcher system. Finally, we show that the cell wall of several Gram-positive bacteria could be functionalized through the external addition of the recombinant engineered Bap-amyloid domain. Overall, this work shows a simple and modulable system for biofilm functionalization based on the facultative protein Bap. © 2022, The Author(s)., This research was supported by grants from the Spanish Ministry of Science and Technology RTI2018-096011-B-I00 to J.V. and PID2020-113494RB-I00 to IL. L.M.-C. was supported by the predoctoral program of the Universidad Pública de Navarra.

Bacteria synchronize the expression of genes with related functions by organizing genes into operons so that they are cotranscribed
together in a single polycistronic messenger RNA. However, some cellular processes may benefit if the simultaneous production of the
operon proteins coincides with the inhibition of the expression of an antagonist gene. To coordinate such situations, bacteria have
evolved noncontiguous operons (NcOs), a subtype of operons that contain one or more genes that are transcribed in the opposite
direction to the other operon genes. This structure results in overlapping transcripts whose expression is mutually repressed. The
presence of NcOs cannot be predicted computationally and their identification requires a detailed knowledge of the bacterial transcriptome. In this study, we used direct RNA sequencing methodology to determine the NcOs map in the Staphylococcus aureus genome.
We detected the presence of 18 NcOs in the genome of S. aureus and four in the genome of the lysogenic prophage 80α. The identified
NcOs comprise genes involved in energy metabolism, metal acquisition and transport, toxin–antitoxin systems, and control of the
phage life cycle. Using the menaquinone operon as a proof of concept, we show that disarrangement of the NcO architecture results
in a reduction of bacterial fitness due to an increase in menaquinone levels and a decrease in the rate of oxygen consumption. Our
study demonstrates the significance of NcO structures in bacterial physiology and emphasizes the importance of combining operon
maps with transcriptomic data to uncover previously unnoticed functional relationships between neighbouring genes., This work was financially supported by the Spanish Ministry of Science, Innovation and Universities grant PID2020-113494RB-I00/AEI/10.13039/501100011033. (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I.L., and JSPS KAKENHI grant number JP23H02719 and the Institute for Fermentation, Osaka to H.H, respectively. P. I-S. was supported by a F.P.I. (PRE2021-097385) contract from the Spanish Ministry of Science, Innovation and Universities. A.S.M. was supported by a contract from the University Department of Navarra government (Res. 229E/2020 Microbiomics grant). Open access funding provided by Universidad Publica de Navarra.

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI). Surface adhesins play an important
role in the primary attachment to plasma proteins that coat the surface of prosthetic devices after implantation.
Previous efforts to identify a genetic component of the bacterium that confers an enhanced capacity to cause PJI
have focused on gene content, kmers, or single-nucleotide polymorphisms (SNPs) in coding sequences. Here,
using a collection of S. aureus strains isolated from PJI and wounds, we investigated whether genetic variations in
the regulatory region of genes encoding surface adhesins lead to differences in their expression levels and
modulate the capacity of S. aureus to colonize implanted prosthetic devices. The data revealed that S. aureus
isolates from the same clonal complex (CC) contain a specific pattern of SNPs in the regulatory region of genes
encoding surface adhesins. As a consequence, each clonal lineage shows a specific profile of surface proteins
expression. Co-infection experiments with representative isolates of the most prevalent CCs demonstrated that
some lineages have a higher capacity to colonize implanted catheters in a murine infection model, which
correlated with a greater ability to form a biofilm on coated surfaces with plasma proteins. Together, results
indicate that differences in the expression level of surface adhesins may modulate the propensity of S. aureus
strains to cause PJI. Given the high conservation of surface proteins among staphylococci, our work lays the
framework for investigating how diversification at intergenic regulatory regions affects the capacity of S. aureus
to colonize the surface of medical implants., This work was financially supported by the Spanish Ministry of Science and Innovation grant PID2020-113494RB-I00 to I.L. (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union), the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 754412 [MoRE2020 - Region Väs-tra Götaland], and the IngaBritt and Arne Lundberg Foundation (LU2021-0048). L.M.L was supported by the European Union's H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 801586 (IberusTalent).

Bacteria synchronize the expression of genes with related functions by organizing genes into operons so that they are cotranscribed together in a single polycistronic messenger RNA. However, some cellular processes may benefit if the simultaneous production of the operon proteins coincides with the inhibition of the expression of an antagonist gene. To coordinate such situations, bacteria have evolved noncontiguous operons (NcOs), a subtype of operons that contain one or more genes that are transcribed in the opposite direction to the other operon genes. This structure results in overlapping transcripts whose expression is mutually repressed. The presence of NcOs cannot be predicted computationally and their identification requires a detailed knowledge of the bacterial transcriptome. In this study, we used direct RNA sequencing methodology to determine the NcOs map in the Staphylococcus aureus genome. We detected the presence of 18 NcOs in the genome of S. aureus and four in the genome of the lysogenic prophage 80α. The identified NcOs comprise genes involved in energy metabolism, metal acquisition and transport, toxin–antitoxin systems, and control of the phage life cycle. Using the menaquinone operon as a proof of concept, we show that disarrangement of the NcO architecture results in a reduction of bacterial fitness due to an increase in menaquinone levels and a decrease in the rate of oxygen consumption. Our study demonstrates the significance of NcO structures in bacterial physiology and emphasizes the importance of combining operon maps with transcriptomic data to uncover previously unnoticed functional relationships between neighbouring genes., This work was financially supported by the Spanish Ministry of Science, Innovation and Universities grant PID2020-113494RB-I00/AEI/10.13039/501100011033. (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I.L., and JSPS KAKENHI grant number JP23H02719 and the Institute for Fermentation, Osaka to H.H, respectively. P. I-S. was supported by a F.P.I. (PRE2021-097385) contract from the Spanish Ministry of Science, Innovation and Universities. A.S.M. was supported by a contract from the University Department of Navarra government (Res. 229E/2020 Microbiomics grant). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Open access funding provided by Universidad Publica de Navarra., Peer Reviewed