CONICET | Buscador de Institutos y Recursos Humanos

Group II introns are self-splicing RNAs widely distributed among bacteria. These ribozymes code for its cofactor, -the intron encoded protein (IEP)- that mainly has a reverse transcriptase, a maturase, and an endonuclease activities. Most bacterial introns are associated to mobile elements, such as conjugative plasmids and transposons that promote horizontal transfer events (HT). The S.ma.I2 group II intron is a plasmid-borne element found in Serratia marcescens. Evolutionary analysis suggests that this intron has been transferred from marine environments to clinical niches. Little is known about the impact this ribozyme exerts over a bacterial host after an HT event. The aim of this work was to evaluate the effect caused by S.ma.I2 RNA at high cellular concentrations. We cloned theS.ma.I2 group II intron is a plasmid-borne element found in Serratia marcescens. Evolutionary analysis suggests that this intron has been transferred from marine environments to clinical niches. Little is known about the impact this ribozyme exerts over a bacterial host after an HT event. The aim of this work was to evaluate the effect caused by S.ma.I2 RNA at high cellular concentrations. We cloned theSerratia marcescens. Evolutionary analysis suggests that this intron has been transferred from marine environments to clinical niches. Little is known about the impact this ribozyme exerts over a bacterial host after an HT event. The aim of this work was to evaluate the effect caused by S.ma.I2 RNA at high cellular concentrations. We cloned theS.ma.I2 RNA at high cellular concentrations. We cloned the S.ma.I2 along with its exons (1000 bp) in the pCR2.1 vector downstream of the T7 RNA polymerase promoter and introduced the plasmid in E. coli BL21 (DE3). Our results showed that background levels of S.ma.I2 intron transcription (0 mM IPTG) caused by the T7 promoter hindered bacterial growth. Upon induction with increasing concentrations of IPTG (0.1 to 1 mM), we were able to significantly inhibit cell growth. Then, we engineered two constructs, the first containing S.ma.I2 with only 100bp of surrounding exons (pCR-SmaI2), and the second carrying solely the iep gene (pCR-IEP). A mild effect on cell growth was observed for pCR-IEP whereas pCR-SmaI2 maintained growth inhibition. Further, we evaluated the stability and maintenance of plasmids expressing the S.ma.I2 intron by serial growth of independent replicates (n=6) followed by plasmid extraction. Faster growing replicates showed a modification in the plasmid profile correlating with the loss of a fragment. Taken together, our results show that the basal expression of the S.ma.I2 intron in E. coli BL21 (DE3) has a direct effect on bacterial growth, which can be reduced by increasing concentrations of IPTG. The S.ma.I2 RNA instead of the protein IEP is most likely responsible for this inhibition, and consequently compromises plasmid stability.I2 along with its exons (1000 bp) in the pCR2.1 vector downstream of the T7 RNA polymerase promoter and introduced the plasmid in E. coli BL21 (DE3). Our results showed that background levels of S.ma.I2 intron transcription (0 mM IPTG) caused by the T7 promoter hindered bacterial growth. Upon induction with increasing concentrations of IPTG (0.1 to 1 mM), we were able to significantly inhibit cell growth. Then, we engineered two constructs, the first containing S.ma.I2 with only 100bp of surrounding exons (pCR-SmaI2), and the second carrying solely the iep gene (pCR-IEP). A mild effect on cell growth was observed for pCR-IEP whereas pCR-SmaI2 maintained growth inhibition. Further, we evaluated the stability and maintenance of plasmids expressing the S.ma.I2 intron by serial growth of independent replicates (n=6) followed by plasmid extraction. Faster growing replicates showed a modification in the plasmid profile correlating with the loss of a fragment. Taken together, our results show that the basal expression of the S.ma.I2 intron in E. coli BL21 (DE3) has a direct effect on bacterial growth, which can be reduced by increasing concentrations of IPTG. The S.ma.I2 RNA instead of the protein IEP is most likely responsible for this inhibition, and consequently compromises plasmid stability.E. coli BL21 (DE3). Our results showed that background levels of S.ma.I2 intron transcription (0 mM IPTG) caused by the T7 promoter hindered bacterial growth. Upon induction with increasing concentrations of IPTG (0.1 to 1 mM), we were able to significantly inhibit cell growth. Then, we engineered two constructs, the first containing S.ma.I2 with only 100bp of surrounding exons (pCR-SmaI2), and the second carrying solely the iep gene (pCR-IEP). A mild effect on cell growth was observed for pCR-IEP whereas pCR-SmaI2 maintained growth inhibition. Further, we evaluated the stability and maintenance of plasmids expressing the S.ma.I2 intron by serial growth of independent replicates (n=6) followed by plasmid extraction. Faster growing replicates showed a modification in the plasmid profile correlating with the loss of a fragment. Taken together, our results show that the basal expression of the S.ma.I2 intron in E. coli BL21 (DE3) has a direct effect on bacterial growth, which can be reduced by increasing concentrations of IPTG. The S.ma.I2 RNA instead of the protein IEP is most likely responsible for this inhibition, and consequently compromises plasmid stability.S.ma.I2 intron transcription (0 mM IPTG) caused by the T7 promoter hindered bacterial growth. Upon induction with increasing concentrations of IPTG (0.1 to 1 mM), we were able to significantly inhibit cell growth. Then, we engineered two constructs, the first containing S.ma.I2 with only 100bp of surrounding exons (pCR-SmaI2), and the second carrying solely the iep gene (pCR-IEP). A mild effect on cell growth was observed for pCR-IEP whereas pCR-SmaI2 maintained growth inhibition. Further, we evaluated the stability and maintenance of plasmids expressing the S.ma.I2 intron by serial growth of independent replicates (n=6) followed by plasmid extraction. Faster growing replicates showed a modification in the plasmid profile correlating with the loss of a fragment. Taken together, our results show that the basal expression of the S.ma.I2 intron in E. coli BL21 (DE3) has a direct effect on bacterial growth, which can be reduced by increasing concentrations of IPTG. The S.ma.I2 RNA instead of the protein IEP is most likely responsible for this inhibition, and consequently compromises plasmid stability.S.ma.I2 with only 100bp of surrounding exons (pCR-SmaI2), and the second carrying solely the iep gene (pCR-IEP). A mild effect on cell growth was observed for pCR-IEP whereas pCR-SmaI2 maintained growth inhibition. Further, we evaluated the stability and maintenance of plasmids expressing the S.ma.I2 intron by serial growth of independent replicates (n=6) followed by plasmid extraction. Faster growing replicates showed a modification in the plasmid profile correlating with the loss of a fragment. Taken together, our results show that the basal expression of the S.ma.I2 intron in E. coli BL21 (DE3) has a direct effect on bacterial growth, which can be reduced by increasing concentrations of IPTG. The S.ma.I2 RNA instead of the protein IEP is most likely responsible for this inhibition, and consequently compromises plasmid stability.iep gene (pCR-IEP). A mild effect on cell growth was observed for pCR-IEP whereas pCR-SmaI2 maintained growth inhibition. Further, we evaluated the stability and maintenance of plasmids expressing the S.ma.I2 intron by serial growth of independent replicates (n=6) followed by plasmid extraction. Faster growing replicates showed a modification in the plasmid profile correlating with the loss of a fragment. Taken together, our results show that the basal expression of the S.ma.I2 intron in E. coli BL21 (DE3) has a direct effect on bacterial growth, which can be reduced by increasing concentrations of IPTG. The S.ma.I2 RNA instead of the protein IEP is most likely responsible for this inhibition, and consequently compromises plasmid stability.S.ma.I2 intron by serial growth of independent replicates (n=6) followed by plasmid extraction. Faster growing replicates showed a modification in the plasmid profile correlating with the loss of a fragment. Taken together, our results show that the basal expression of the S.ma.I2 intron in E. coli BL21 (DE3) has a direct effect on bacterial growth, which can be reduced by increasing concentrations of IPTG. The S.ma.I2 RNA instead of the protein IEP is most likely responsible for this inhibition, and consequently compromises plasmid stability.S.ma.I2 intron in E. coli BL21 (DE3) has a direct effect on bacterial growth, which can be reduced by increasing concentrations of IPTG. The S.ma.I2 RNA instead of the protein IEP is most likely responsible for this inhibition, and consequently compromises plasmid stability.S.ma.I2 RNA instead of the protein IEP is most likely responsible for this inhibition, and consequently compromises plasmid stability.