Open Access
Noncontiguous operon atlas for the Staphylococcus aureus genome
(Oxford University Press, 2024) Iturbe Sanz, Pablo; San Martín Bernal, Álvaro; Hamamoto, Hiroshi; Marcet Houben, Marina; Galbaldón, Toni; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
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.
Open Access
Fluorescent molecular beacons mimicking RNA secondary structures to study RNA chaperone activity
(Humana Press, 2020) Menéndez Gil, Pilar; Caballero Sánchez, Carlos; Solano Goñi, Cristina; Toledo Arana, Alejandro; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5′ and 3′ ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases.
Open Access
Elevated c-di-GMP levels promote biofilm formation and biodesulfurization capacity of Rhodococcus erythropolis
(Society for Applied Microbiology, 2020) Dorado Morales, Pedro; Martínez, Igor; Rivero Buceta, Virginia; Díaz, Eduardo; Bähre, Heike; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; Ciencias de la Salud; Osasun Zientziak
Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di-GMP (c-di-GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels.
Open Access
Experimental polymorphism survey in intergenic regions of the icaADBCR locus in Staphylococcus aureus isolates from periprosthetic joint infections
(MDPI, 2022) Morales Laverde, Liliana Andrea; Echeverz Sarasúa, Maite; Trobos, Margarita; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak
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.
Open Access
Regulation of gene expression by non-phosphorylated response regulators
(Institut d'Estudis Catalans, 2021) Gómez Arrebola, Carmen; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak
Two-component systems (TCSs) are a prominent sensory system in bacteria. A prototypical TCS comprises a membrane-bound sensor histidine kinase (HK) responsible for sensing the signal and a cytoplasmic response regulator (RR) that controls target gene expression. Signal binding activates a phosphotransfer cascade from the HK to the RR. As a result, the phosphorylated RR undergoes a conformational change that leads to activation of the response. Growing experimental evidence indicates that unphosphorylated RRs may also have regulatory functions, and thus, the classical view that the RR is only active when it is phosphorylated needs to be revisited. In this review, we highlight the most recent findings showing that RRs in the non-phosphorylated state control critical bacterial processes that range from secretion of factors to the host, antibiotic resistance, iron transport, stress response, and cell-wall metabolism to biofilm development.
Open Access
A DIVA vaccine strain lacking RpoS and the secondary messenger c-di-GMP for protection against salmonellosis in pigs
(BioMed Central, 2020) Gil Puig, Carmen; Latasa Osta, Cristina; García Ona, Enrique; Lázaro, Isidro; Labairu, Javier; Echeverz Sarasúa, Maite; Burgui Erice, Saioa; García Martínez, Begoña; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako Gobernua, IIM 13329.RI1
Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA).
Open Access
Staphylococcus aureus susceptibility to complestatin and corbomycin depends on the VraSR two-component system
(American Society for Microbiology, 2023) Gómez Arrebola, Carmen; Hernández, Sara B.; Culp, Elizabeth J.; Wright, Gerard D.; Solano Goñi, Cristina; Cava, Felipe; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
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.
Open Access
The impact of two-component sensorial network in staphylococcal speciation
(Elsevier, 2020) Rapún Araiz, Beatriz; Haag, Andreas F.; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak
Bacteria use two-component systems (TCSs) to sense and respond to their environments. Free-living bacteria usually contain dozens of TCSs, each of them responsible for sensing and responding to a different range of signals. Differences in the content of two-component systems are related with the capacity of the bacteria to colonize different niches or improve the efficiency to grow under the conditions of the existing niche. This review highlights differences in the TCS content between Staphylococcus aureus and Staphylococcus saprophyticus as a case study to exemplify how the ability to sense and respond to the environment is relevant for bacterial capacity to colonize and survive in/on different body surfaces.
Open Access
Functional analysis of intergenic regulatory regions of genes encoding surface adhesins in Staphylococcus aureus isolates from periprosthetic joint infections
(Elsevier, 2022) Morales Laverde, Liliana Andrea; Trobos, Margarita; Echeverz Sarasúa, Maite; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak
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.
Open Access
Fitness cost evolution of natural plasmids of staphylococcus aureus
(American Society for Microbiology, 2021) Dorado Morales, Pedro; Garcillán-Barcia, María Pilar; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; Ciencias de la Salud; Osasun Zientziak
Plasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids.