Genome-wide identification of tolerance mechanisms toward p-coumaric acid in Pseudomonas putida.

The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p-coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome-wide approach, Tn-seq. Libraries containing a large number of miniTn5-Km transposon insertion mutants were grown in the presence and absence of p-coumaric acid, and the enrichment or depletion of mutants was quantified by high-throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p-coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p-coumaric acid is related to transport and membrane integrity.

Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida.

Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings.IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization.

Genome-wide mapping of transcription start sites yields novel insights into the primary transcriptome of Pseudomonas putida.

The environmental bacterium Pseudomonas putida is an organism endowed with a versatile metabolism and stress tolerance traits that are desirable in an efficient production organism. In this work, differential RNA sequencing was used to investigate the primary transcriptome and RNA regulatory elements of P. putida strain KT2440. A total of 7937 putative transcription start sites (TSSs) were identified, where over two-thirds were located either on the opposite strand or internal to annotated genes. For TSSs associated with mRNAs, sequence analysis revealed a clear Shine-Dalgarno sequence but a lack of conserved overrepresented promoter motifs. These TSSs defined approximately 50 leaderless transcripts and an abundance of mRNAs with long leader regions of which 18 contain RNA regulatory elements from the Rfam database. The thiamine pyrophosphate riboswitch upstream of the thiC gene was examined using an in vivo assay with GFP-fusion vectors and shown to function via a translational repression mechanism. Furthermore, 56 novel intergenic small RNAs and 8 putative actuaton transcripts were detected, as well as 8 novel open reading frames (ORFs). This study illustrates how global mapping of TSSs can yield novel insights into the transcriptional features and RNA output of bacterial genomes.

Differential expression of small RNAs under chemical stress and fed-batch fermentation in E. coli.

BACKGROUND: Bacterial small RNAs (sRNAs) are recognized as posttranscriptional regulators involved in the control of bacterial lifestyle and adaptation to stressful conditions. Although chemical stress due to the toxicity of precursor and product compounds is frequently encountered in microbial bioprocessing applications, the involvement of sRNAs in this process is not well understood. We have used RNA sequencing to map sRNA expression in E. coli under chemical stress and high cell density fermentation conditions with the aim of identifying sRNAs involved in the transcriptional response and those with potential roles in stress tolerance. RESULTS: RNA sequencing libraries were prepared from RNA isolated from E. coli K-12 MG1655 cells grown under high cell density fermentation conditions or subjected to chemical stress with twelve compounds including four organic solvent-like compounds, four organic acids, two amino acids, geraniol and decanoic acid. We have discovered 253 novel intergenic transcripts with this approach, adding to the roughly 200 intergenic sRNAs previously reported in E. coli. There are eighty-four differentially expressed sRNAs during fermentation, of which the majority are novel, supporting possible regulatory roles for these transcripts in adaptation during different fermentation stages. There are a total of 139 differentially expressed sRNAs under chemical stress conditions, where twenty-nine exhibit significant expression changes in multiple tested conditions, suggesting that they may be involved in a more general chemical stress response. Among those with known functions are sRNAs involved in regulation of outer membrane proteins, iron availability, maintaining envelope homeostasis, as well as sRNAs incorporated into complex networks controlling motility and biofilm formation. CONCLUSIONS: This study has used deep sequencing to reveal a wealth of hitherto undescribed sRNAs in E. coli and provides an atlas of sRNA expression during seventeen different growth and stress conditions. Although the number of novel sRNAs with regulatory functions is unknown, several exhibit specific expression patterns during high cell density fermentation and are differentially expressed in the presence of multiple chemicals, suggesting they may play regulatory roles during these stress conditions. These novel sRNAs, together with specific known sRNAs, are candidates for improving stress tolerance and our understanding of the E. coli regulatory network during fed-batch fermentation.

RNAi as a Tool to Study Virulence in the Pathogenic Yeast Candida glabrata.

The yeast Candida glabrata is a major opportunistic pathogen causing mucosal and systemic infections in humans. Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to this yeast's intrinsic and frequently adapting antifungal resistance. To understand and treat C. glabrata infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance. We established an RNA interference (RNAi) system in C. glabrata by expressing the Dicer and Argonaute genes from Saccharomyces castellii (a budding yeast with natural RNAi). Our experiments with reporter genes and putative virulence genes showed that the introduction of RNAi resulted in 30 and 70% gene-knockdown for the construct-types antisense and hairpin, respectively. The resulting C. glabrata RNAi strain was used for the screening of a gene library for new virulence-related genes. Phenotypic profiling with a high-resolution quantification of growth identified genes involved in the maintenance of cell integrity, antifungal drugs, and ROS resistance. The genes identified by this approach are promising targets for the treatment of C. glabrata infections.