Open Access
Genome-wide antisense transcription drives mRNA processing in bacteria
(National Academy of Sciences, 2011) Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; Dobin, Alexander; Villanueva San Martín, Maite; Ruiz de los Mozos Aliaga, Igor; Vergara Irigaray, Marta; Segura, Víctor; Fagegaltier, Delphine; Penadés, José R.; Valle Turrillas, Jaione; Solano Goñi, Cristina; Gingeras, Thomas R.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
RNA deep sequencing technologies are revealing unexpected levels of complexity in bacterial transcriptomes with the discovery of abundant noncoding RNAs, antisense RNAs, long 5′ and 3′ untranslated regions, and alternative operon structures. Here, by applying deep RNA sequencing to both the long and short RNA fractions (<50 nucleotides) obtained from the major human pathogen Staphylococcus aureus, we have detected a collection of short RNAs that is generated genome-wide through the digestion of overlapping sense/antisense transcripts by RNase III endoribonuclease. At least 75% of sense RNAs from annotated genes are subject to this mechanism of antisense processing. Removal of RNase III activity reduces the amount of short RNAs and is accompanied by the accumulation of discrete antisense transcripts. These results suggest the production of pervasive but hidden antisense transcription used to process sense transcripts by means of creating double-stranded substrates. This process of RNase III-mediated digestion of overlapping transcripts can be observed in several evolutionarily diverse Gram-positive bacteria and is capable of providing a unique genome-wide posttranscriptional mechanism to adjust mRNA levels.
Open Access
Base pairing interaction between 5′- and 3′-UTRs controls icaR mRNA translation in Staphylococcus aureus
(Public Library of Science, 2013) Ruiz de los Mozos Aliaga, Igor; Vergara Irigaray, Marta; Segura, Víctor; Villanueva San Martín, Maite; Bitarte Manzanal, Nerea; Saramago, Margarida; Domingues, Susana; Arraiano, Cecilia M.; Fechter, Pierre; Romby, Pascale; Valle Turrillas, Jaione; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The presence of regulatory sequences in the 39 untranslated region (39-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 39-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the major human pathogen Staphylococcus aureus carry 39-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 39-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 39-UTRs may play in controlling mRNA expression. We showed that base pairing between the 39- UTR and the Shine-Dalgarno (SD) region of icaR mRNA interferes with the translation initiation complex and generates a double-stranded substrate for RNase III. Deletion or substitution of the motif (UCCCCUG) within icaR 39-UTR was sufficient to abolish this interaction and resulted in the accumulation of IcaR repressor and inhibition of biofilm development. Our findings provide a singular example of a new potential post-transcriptional regulatory mechanism to modulate bacterial gene expression through the interaction of a 39-UTR with the 59-UTR of the same mRNA.
Open Access
A strain of Bacillus thuringiensis containing a novel cry7Aa2 gene that is toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)
(MDPI, 2019) Domínguez Arrizabalaga, Mikel; Villanueva San Martín, Maite; Fernández González, Ana Beatriz; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua
The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity compared to Cry7Aa1 and a predicted molecular mass of 129.4 kDa. The primary structure of Cry7Aa2, which had eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that after in vitro trypsin incubation, the protein was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae, one major proteinase-resistant fragment of slightly smaller size was produced. The spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 value of 18.8 mu g/mL, which was statistically similar to the estimated LC50 of 20.8 mu g/mL for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 3.8-fold to LC50 = 4.9 mu g/mL. The potential advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.
Open Access
Quantification of dose-mortality responses in adult Diptera: validation using Ceratitis capitata and Drosophila suzukii responses to spinosad
(Public Library of Science, 2019) Valtierra de Luis, Daniel; Villanueva San Martín, Maite; Caballero Sánchez, Javier; Matas Casado, Isabel María; Williams, Trevor; Caballero Murillo, Primitivo; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB; Agronomía, Biotecnología y Alimentación; Gobierno de Navarra / Nafarroako Gobernua, BTMOL-PI028; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Quantitative laboratory bioassay methods are required to evaluate the toxicity of novel insecticidal compounds for pest control and to determine the presence of resistance traits. We used a radioactive tracer based on P-32-ATP to estimate the volume of a droplet ingested by two dipteran pests: Ceratitis capitata (Tephritidae) and Drosophila suzukii (Drosophilidae). Using blue food dye it was possible to distinguish between individuals that ingested the solution from those that did not. The average volume ingested by C. capitata adults was 1.968 mu l. Females ingested a similar to 20% greater volume of solution than males. Adults of D. suzukii ingested an average of 0.879 mu l and females ingested similar to 30% greater volume than males. The droplet feeding method was validated using the naturally-derived insecticide spinosad as the active ingredient (a.i.). For C. capitata, the concentration-mortality response did not differ between the sexes or among three different batches of insects. Lethal dose values were calculated based on mean ingested volumes. For C. capitata LD50 values were 1.462 and 1.502 ng a.i./insect for males and females, respectively, equivalent to 0.274 and 0.271 ng a.i./mg for males and females, respectively, when sex-specific variation in body weight was considered. Using the same process for D. suzukii, the LD50 value was estimated at 2.927 ng a.i./insect, or 1.994 ng a.i./mg based on a mean body weight of 1.67 mg for both sexes together. We conclude that this technique could be readily employed for determination of the resistance status and dose-mortality responses of insecticidal compounds in many species of pestiferous Diptera.
Open Access
Potential of Cry10Aa and Cyt2Ba, two minority δ-endotoxins produced by Bacillus thuringiensis ser. israelensis, for the control of Aedes aegypti larvae
(MDPI, 2020) Valtierra de Luis, Daniel; Villanueva San Martín, Maite; Lai, Liliana; Williams, Trevor; Caballero Murillo, Primitivo; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB; Agronomía, Biotecnología y Alimentación
Bacillus thuringiensis ser. israelensis (Bti) has been widely used as microbial larvicide for the control of many species of mosquitoes and blackflies. The larvicidal activity of Bti resides in Cry and Cyt δ-endotoxins present in the parasporal crystal of this pathogen. The insecticidal activity of the crystal is higher than the activities of the individual toxins, which is likely due to synergistic interactions among the crystal component proteins, particularly those involving Cyt1Aa. In the present study, Cry10Aa and Cyt2Ba were cloned fromthe commercial larvicideVectoBac-12AS® and expressed in the acrystalliferous Bt strain BMB171 under the cyt1Aa strong promoter of the pSTAB vector. The LC50 values for Aedes aegypti second instar larvae estimated at 24 hpi for these two recombinant proteins (Cry10Aa and Cyt2Ba) were 299.62 and 279.37 ng/mL, respectively. Remarkable synergistic mosquitocidal activity was observed between Cry10Aa and Cyt2Ba (synergistic potentiation of 68.6-fold) when spore + crystal preparations, comprising a mixture of both recombinant strains in equal relative concentrations, were ingested by A. aegypti larvae. This synergistic activity is among the most powerful described so far with Bt toxins and is comparable to that reported for Cyt1A when interacting with Cry4Aa, Cry4Ba or Cry11Aa. Synergistic mosquitocidal activity was also observed between the recombinant proteins Cyt2Ba and Cry4Aa, but in this case, the synergistic potentiation was 4.6-fold. In conclusion, although Cry10Aa and Cyt2Ba are rarely detectable or appear as minor components in the crystals of Bti strains, they represent toxicity factors with a high potential for the control of mosquito populations.
Open Access
Genetic reductionist approach for studing the two-component signaling system in Staphylococcus aureus
(2014) Villanueva San Martín, Maite; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; Producción Agraria; Nekazaritza Ekoizpena
Staphylococcus aureus es una bacteria ubicua capaz de colonizar una gran variedad de ambientes. En el hombre, S. aureus coloniza las fosas nasales, piel de las axilas, ingles, garganta o incluso el tracto intestinal. Se calcula que un 20% de las personas adultas son portadores nasales de S. aureus. En determinadas circunstancias, la bacteria es capaz de atravesar la barrera epitelial y alcanzar los órganos internos. Cuando esto ocurre, S. aureus se convierte en un patógeno muy versátil capaz de causar enfermedades muy diversas, que pueden ir desde infecciones leves como forúnculos o abscesos hasta enfermedades graves como endocarditis, osteomielitis, neumonía o síndrome del shock tóxico. El desarrollo de S. aureus en distintos ambientes requiere que la bacteria sea capaz de sensar las condiciones ambientales, transmitir los estímulos al citoplasma y activar los cambios necesarios para adecuar la fisiología a dicho ambiente. El principal mecanismo para sensar y responder a las señales ambientales en bacterias son los sistemas de dos-componentes (TCSs). Los TCSs están formados por un sensor de membrana o histidinekinase (HK) y un regulador de respuesta citoplásmico (RR). En el proceso de activación, el sensor recibe su señal específica y se auto fosforila en un dominio histidina. A continuación el fosfato es transferido al residuo aspártico del RR que se encuentra en el citoplasma. De esta forma, el RR se activa y desencadena una respuesta que será acorde a la señal recibida. Normalmente, una bacteria posee varios TCSs, siendo su número proporcional al tamaño del genoma, al número de ambientes distintos en las que es capaz de crecer y a la complejidad de su diferenciación celular. Así, bacterias que viven en ambientes muy constantes, como las bacterias intracelulares estrictas carecen de TCSs, mientras que bacterias que viven en ambientes diversos pueden poseer cientos de ellos. En relación con el número y la función de los TCSs existen varias preguntas que hasta ahora no han sido analizadas: ¿cuántos TCSs necesita una bacteria de vida libre? ¿Son necesarios los TCSs cuando la bacteria crece en un ambiente constante? ¿Existe activación cruzada entre TCSs distintos in vivo? Para responder a estas preguntas y realizar un estudio global de los procesos celulares controlados por los TCSs, en esta tesis hemos realizado una aproximación genética reduccionista usando como modelo dos cepas genéticamente no relacionadas de S. aureus. El trabajo ha consistido en la deleción completa de los 15 TCSs no esenciales que posee S. aureus y la mutación del sensor (WalK) del TCS walKR, cuya deleción completa resulta letal. Las bacterias resultantes carecen del sistema sensorial y su obtención demuestra que en condiciones ambientales constantes estos sistemas son dispensables para la vida de S. aureus. Los mutantes deficientes en los TCSs muestran niveles de crecimiento indistinguibles a los de la cepa salvaje a 37ºC y 44ºC y un patrón metabólico similar. En cambio, los mutantes tienen deficiencias en el crecimiento a 28ºC, pierden la capacidad de reducción de nitratos, muestran mayor sensibilidad al Tritón X-100 así como una menor capacidad para sobrevivir en el ambiente e invadir células. Así mismo, los mutantes tienen reducida su virulencia y capacidad de colonizar órganos en un modelo de infección de ratón. Todos los fenotipos del mutante deficiente en los TCSs podían ser restaurados por la expresión ectópica de un único TCS indicando que cada uno de los fenotipos depende de un único TCS. Finalmente, la cepa deficiente en los TCSs ha sido utilizada como una plataforma para el estudio de la especificidad de transmisión de señal in vivo, un concepto que en inglés se denomina ‘cross-talk’ y que hasta ahora había sido estudiada in vitro. Para ello, hemos establecido una sencilla metodología que consiste en la complementación del mutante deficiente en TCSs con una colección de plásmidos que contienen una combinación de la familia de HKs y un RR. El análisis de las cepas complementadas nos ha permitido identificar la existencia de activación cruzada entre GraS y ArlR. Esta activación cruzada tiene lugar incluso en presencia de sus correspondientes parejas, la HK ArlS y el RR GraR. Teniendo en cuenta que durante este análisis global sólo hemos detectado activación cruzada entre estos TCSs, la conclusión de nuestro estudio es que la activación cruzada entre los TCSs puede ocurrir in vivo, pero no es frecuente. En el futuro las cepas deficientes en los TCSs, o cepas derivadas conteniendo únicamente uno de ellos, servirán para identificar el regulón que controla cada TCS o para identificar nuevos fármacos que bloqueen específicamente a los TCSs.
Open Access
Sensory deprivation in Staphylococcus aureus
(Springer Nature, 2018) Villanueva San Martín, Maite; García Martínez, Begoña; Valle Turrillas, Jaione; Rapún Araiz, Beatriz; Ruiz de los Mozos Aliaga, Igor; Solano Goñi, Cristina; Martí, Miguel; Penadés, José R.; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Bacteria use two-component systems (TCSs) to sense and respond to environmental changes. The core genome of the major human pathogen Staphylococcus aureus encodes 16 TCSs, one of which (WalRK) is essential. Here we show that S. aureus can be deprived of its complete sensorial TCS network and still survive under growth arrest conditions similarly to wild-type bacteria. Under replicating conditions, however, the WalRK system is necessary and sufficient to maintain bacterial growth, indicating that sensing through TCSs is mostly dispensable for living under constant environmental conditions. Characterization of S. aureus derivatives containing individual TCSs reveals that each TCS appears to be autonomous and self-sufficient to sense and respond to specific environmental cues, although some level of cross-regulation between non-cognate sensor-response regulator pairs occurs in vivo. This organization, if confirmed in other bacterial species, may provide a general evolutionarily mechanism for flexible bacterial adaptation to life in new niches.
Open Access
Regulation of heterogenous lexA expression in staphylococcus aureus by an antisense RNA originating from transcriptional read-through upon natural mispairings in the sbrB intrinsic terminator
(MDPI, 2022) Bastet, Laurène; Bustos-Sanmamed, Pilar; Catalán Moreno, Arancha; Caballero Sánchez, Carlos; Cuesta Ferre, Sergio; Matilla Cuenca, Leticia; Villanueva San Martín, Maite; Valle Turrillas, Jaione; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.
Open Access
Insecticidal activity of bacillus thuringiensis proteins against coleopteran pests
(MDPI, 2020) Domínguez Arrizabalaga, Mikel; Villanueva San Martín, Maite; Escriche, Baltasar; Ancín Azpilicueta, Carmen; Caballero Murillo, Primitivo; Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB; Ciencias
Bacillus thuringiensis is the most successful microbial insecticide agent and its proteins have been studied for many years due to its toxicity against insects mainly belonging to the orders Lepidoptera, Diptera and Coleoptera, which are pests of agro-forestry and medical-veterinary interest. However, studies on the interactions between this bacterium and the insect species classified in the order Coleoptera are more limited when compared to other insect orders. To date, 45 Cry proteins, 2 Cyt proteins, 11 Vip proteins, and 2 Sip proteins have been reported with activity against coleopteran species. A number of these proteins have been successfully used in some insecticidal formulations and in the construction of transgenic crops to provide protection against main beetle pests. In this review, we provide an update on the activity of Bt toxins against coleopteran insects, as well as specific information about the structure and mode of action of coleopteran Bt proteins.
Open Access
The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus
(Oxford University Press, 2018) Caballero Sánchez, Carlos; Menéndez Gil, Pilar; Catalán Moreno, Arancha; Vergara Irigaray, Marta; García Martínez, Begoña; Segura, Víctor; Irurzun Domínguez, Naiara; Villanueva San Martín, Maite; Ruiz de los Mozos Aliaga, Igor; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
RNA-binding proteins (RBPs) are essential to finetune gene expression. RBPs containing the coldshock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBPimmunoprecipitation- microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA posttranscriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5 UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5 UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist.