NMR-Metabolomics Shows That BolA Is an Important Modulator of Salmonella Typhimurium Metabolic Processes under Virulence Conditions.

BolA is a ubiquitous global transcription factor. Despite its clear role in the induction of important stress-resistant physiological changes and its recent implication in the virulence of Salmonella, further research is required to shed light on the pathways modulated by BolA. In this study, we resorted to untargeted 1H-NMR metabolomics to understand the impact of BolA on the metabolic profile of Salmonella Typhimurium, under virulence conditions. Three strains of S. Typhimurium SL1344 were studied: An SL1344 strain transformed with an empty plasmid (control), a bolA knockout mutant (ΔbolA), and a strain overexpressing bolA (bolA+). These strains were grown in a minimal virulence-inducing medium and cells were collected at the end of the exponential and stationary phases. The extracts were analyzed by NMR, and multivariate and univariate statistical analysis were performed to identify significant alterations. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of 1H-NMR data allowed the discrimination between the metabolic profiles of these strains, revealing increased levels of acetate, valine, alanine, NAD+, succinate, coenzyme A, glutathione, and putrescine in bolA+. These results indicate that BolA regulates pathways related to stress resistance and virulence, being an important modulator of the metabolic processes needed for S. Typhimurium infection.

Stress Response Protein BolA Influences Fitness and Promotes Salmonella enterica Serovar Typhimurium Virulence.

The intracellular pathogen Salmonella enterica serovar Typhimurium has emerged as a major cause of foodborne illness, representing a severe clinical and economic concern worldwide. The capacity of this pathogen to efficiently infect and survive inside the host depends on its ability to synchronize a complex network of virulence mechanisms. Therefore, the identification of new virulence determinants has become of paramount importance in the search of new targets for drug development. BolA-like proteins are widely conserved in all kingdoms of life. In Escherichia coli, this transcription factor has a critical regulatory role in several mechanisms that are tightly related to bacterial virulence. Therefore, in the present work we used the well-established infection model Galleria mellonella to evaluate the role of BolA protein in S Typhimurium virulence. We have shown that BolA is an important player in S Typhimurium pathogenesis. Specifically, the absence of BolA leads to a defective virulence capacity that is most likely related to the remarkable effect of this protein on S Typhimurium evasion of the cellular response. Furthermore, it was demonstrated that BolA has a critical role in bacterial survival under harsh conditions since BolA conferred protection against acidic and oxidative stress. Hence, we provide evidence that BolA is a determining factor in the ability of Salmonella to survive and overcome host defense mechanisms, and this is an important step in progress to an understanding of the pathways underlying bacterial virulence.IMPORTANCE BolA has been described as an important protein for survival in the late stages of bacterial growth and under harsh environmental conditions. High levels of BolA in stationary phase and under stresses have been connected with a plethora of phenotypes, strongly suggesting its important role as a master regulator. Here, we show that BolA is a determining factor in the ability of Salmonella to survive and overcome host defense mechanisms, and this is an important step in progress to an understanding of the pathways underlying bacterial virulence. This work constitutes a relevant step toward an understanding of the role of BolA protein and may have an important impact on future studies in other organisms. Therefore, this study is of utmost importance for understanding the genetic and molecular bases involved in the regulation of Salmonella virulence and may contribute to future industrial and public health care applications.

BolA Is Required for the Accurate Regulation of c-di-GMP, a Central Player in Biofilm Formation.

The bacterial second messenger cyclic dimeric GMP (c-di-GMP) is a nearly ubiquitous intracellular signaling molecule involved in the transition from the motile to the sessile/biofilm state in bacteria. C-di-GMP regulates various cellular processes, including biofilm formation, motility, and virulence. BolA is a transcription factor that promotes survival in different stresses and is also involved in biofilm formation. Both BolA and c-di-GMP participate in the regulation of motility mechanisms leading to similar phenotypes. Here, we establish the importance of the balance between these two factors for accurate regulation of the transition between the planktonic and sessile lifestyles. This balance is achieved by negative-feedback regulation of BolA and c-di-GMP. BolA not only contributes directly to the motility of bacteria but also regulates the expression of diguanylate cyclases and phosphodiesterases. This expression modulation influences the synthesis and degradation of c-di-GMP, while this signaling metabolite has a negative influence in bolA mRNA transcription. Finally, we present evidence of the dominant role of BolA in biofilm, showing that, even in the presence of elevated c-di-GMP levels, biofilm formation is reduced in the absence of BolA. C-di-GMP is one of the most important bacterial second messengers involved in several cellular processes, including virulence, cell cycle regulation, biofilm formation, and flagellar synthesis. In this study, we unravelled a direct connection between the bolA morphogene and the c-di-GMP signaling molecule. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in Escherichia coli This work provides important clues that can be helpful in the development of new strategies, and the results can be applied to other organisms with relevance for human health.IMPORTANCE Bacterial cells have evolved several mechanisms to cope with environmental stresses. BolA-like proteins are widely conserved from prokaryotes to eukaryotes, and in Escherichia coli, in addition to its pleiotropic effects, this protein plays a determinant role in bacterial motility and biofilm formation regulation. Similarly, the bacterial second messenger c-di-GMP is a molecule with high importance in coordinating the switch between planktonic and sessile life in bacteria. Here we have unravelled the importance of accurate regulation of cross-talk between BolA and c-di-GMP for a proper response in the regulation of these bacterial lifestyles. This finding underlines the complexity of bacterial cell regulation, revealing the existence of one additional tool for fine-tuning such important cellular molecular mechanisms. The relationship between BolA and c-di-GMP gives new perspectives regarding biofilm formation and opens the possibility to extend our studies to other organisms with relevance for human health.

The role of RNase R in trans-translation and ribosomal quality control.

Gene expression not only depends on the rate of transcription but is also largely controlled at the post-transcriptional level. Translation rate and mRNA decay greatly influence the final protein levels. Surveillance mechanisms are essential to ensure the quality of the RNA and proteins produced. Trans-translation is one of the most important systems in the quality control of bacterial translation. This process guarantees the destruction of abnormal proteins and also leads to degradation of the respective defective RNAs through the action of Ribonuclease R (RNase R). This exoribonuclease hydrolyzes RNAs starting from their 3' end. Besides its involvement in trans-translation, RNase R also participates in the quality control of rRNA molecules involved in ribosomal biogenesis. RNase R is thus emerging as a key factor in ensuring translation accuracy. This review focuses on issues related to the quality control of translation, with special emphasis on the role of RNase R.

Synergies between RNA degradation and trans-translation in Streptococcus pneumoniae: cross regulation and co-transcription of RNase R and SmpB.

BACKGROUND: Ribonuclease R (RNase R) is an exoribonuclease that recognizes and degrades a wide range of RNA molecules. It is a stress-induced protein shown to be important for the establishment of virulence in several pathogenic bacteria. RNase R has also been implicated in the trans-translation process. Transfer-messenger RNA (tmRNA/SsrA RNA) and SmpB are the main effectors of trans-translation, an RNA and protein quality control system that resolves challenges associated with stalled ribosomes on non-stop mRNAs. Trans-translation has also been associated with deficiencies in stress-response mechanisms and pathogenicity. RESULTS: In this work we study the expression of RNase R in the human pathogen Streptococcus pneumoniae and analyse the interplay of this enzyme with the main components of the trans-translation machinery (SmpB and tmRNA/SsrA). We show that RNase R is induced after a 37°C to 15°C temperature downshift and that its levels are dependent on SmpB. On the other hand, our results revealed a strong accumulation of the smpB transcript in the absence of RNase R at 15°C. Transcriptional analysis of the S. pneumoniae rnr gene demonstrated that it is co-transcribed with the flanking genes, secG and smpB. Transcription of these genes is driven from a promoter upstream of secG and the transcript is processed to yield mature independent mRNAs. This genetic organization seems to be a common feature of Gram positive bacteria, and the biological significance of this gene cluster is further discussed. CONCLUSIONS: This study unravels an additional contribution of RNase R to the trans-translation system by demonstrating that smpB is regulated by this exoribonuclease. RNase R in turn, is shown to be under the control of SmpB. These proteins are therefore mutually dependent and cross-regulated. The data presented here shed light on the interactions between RNase R, trans-translation and cold-shock response in an important human pathogen.

A new target for an old regulator: H-NS represses transcription of bolA morphogene by direct binding to both promoters.

The Escherichia coli bolA morphogene is very important in adaptation to stationary phase and stress response mechanisms. Genes of this family are widespread in gram negative bacteria and in eukaryotes. The expression of this gene is tightly regulated at transcriptional and post-transcriptional levels and its overexpression is known to induce round cellular morphology. The results presented in this report demonstrate that the H-NS protein, a pleiotropic regulator of gene expression, is a new transcriptional modulator of the bolA gene. In this work we show that and in vivo the levels of bolA are down-regulated by H-NS and in vitro this global regulator interacts directly with the bolA promoter region. Moreover, DNaseI foot-printing experiments mapped the interaction regions of H-NS and bolA and revealed that this global regulator binds not only one but both bolA promoters. We provide a new insight into the bolA regulation network demonstrating that H-NS represses the transcription of this important gene.

The critical role of RNA processing and degradation in the control of gene expression.

The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites with different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher-order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression. RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are >20 different RNases. RNase E is a single-strand-specific endonuclease critical for mRNA decay in E. coli. The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double-stranded RNA and family members are involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay of many different mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.