On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli

We thank Frans Bianchi and Franz Ho for assistance with molecular cloning, Tim Rasmussen for providing the pTRC-MscK plasmid, Andrew Robinson for providing the pBAD-mEos3.2 plasmid, Matthias Heinemann for assistance with the flow cytometry measurements, Paul Schavemaker for performing Smoldyn simulations and Michiel Punter for programming ImageJ plugins for PALM reconstructions and single-particle tracking. We thank Ian Booth for critical reading of the manuscript, and Christoffer Åberg and Matteo Gabba for valuable discussions. The authors would like to thank David Dryden and Marcel Reuter for performing preliminary experiments from which this work has been built. The work was funded by the EU FP7 ITN-network program NICHE.Peer reviewedPublisher PD

Properties of the mechanosensitive channel MscS pore revealed by tryptophan scanning mutagenesis

Funding This work was supported by a Wellcome Trust Programme grant [092552/A/10/Z awarded to I.R.B., S.M., J. H. Naismith (University of St Andrews, St Andrews, U.K.), and S. J. Conway (University of Oxford, Oxford, U.K.)] (T.R. and M.D.E.), by a BBSRC grant (A.R.) [BB/H017917/1 awarded to I.R.B., J. H. Naismith, and O. Schiemann (University of St Andrews)], by a Leverhulme Emeritus Fellowship (EM-2012-060\2), and by a CEMI grant to I.R.B. from the California Institute of Technology. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013 FP7/2007-2011) under Grant PITN-GA-2011-289384 (FP7-PEOPLE-2011-ITN NICHE) (H.G.) (awarded to S.M.).Peer reviewedPublisher PD

Characterization of Escherichia coli strains carrying different rpos alleles.

A bactéria Escherichia coli é encontrada em diversos habitats e deve estar preparada para sobreviver e crescer em condições desfavoráveis. A adaptação da bactéria a diferentes condições obriga-a a controlar a expressão de genes de forma eficiente. Uma das formas primárias de controle de expressão gênica é a competição entre os diversos fatores sigma pela ligação ao cerne da RNA polimerase. d70 é o fator sigma mais abundante e participa da transcrição da maioria dos genes de E. coli, enquanto que dS é o segundo em importância e reconhece promotores de genes relacionados à resposta geral ao estresse. O gene rpoS, que codifica para dS é altamente polimórfico, e adquiri mutações frequentemente. A mutação pontual C®T na posição 97 da ORF de rpoS, resulta em um códon de parada TAG (âmbar). Um Shine-Dalgarno alternativo e um códon de início de tradução na posição 157 dá início a uma proteína RpoS truncada, que é parcialmente funcional. Uma das situações de estresse na qual dS é ativado corresponde à privação de fosfato inorgânico. Porém, na limitação deste nutriente ocorre também a ativação do regulon PHO, cujos genes são predominantemente transcritos por d70. Neste trabalho, o efeito da versão truncada de RpoS sobre a expressão de genes dependentes de d70 (lacZ, phoA e pstS) e de genes dependentes de dS (osmY e proU) foi testado. Foram também realizados ensaios de estresse oxidativo, osmótico e pelo frio. O perfil de atividade parcial descrito para RpoSam pôde ser observado em alguns casos, porém em outros o comportamento deste alelo se assemelhou ao do mutante rpoS nulo. Paralelamente, foi testada também uma cepa de E. coli que carrega a mutação âmbar em rpoS, mas esta é suprimida, resultando na expressão de uma proteína RpoS normal. A proteína RpoS truncada não pôde ser visualizada em immunoblots, provavelmente porque esta é traduzida de forma pouco eficiente a partir do Shine-Dalgarno alternativo. Com o objetivo de incrementar a detecção de RpoS em ensaios de imuno-detecção, foi inserida por recombinação alélica uma etiqueta SPA altamente imunogênica na porção C-terminal da proteína.Escherichia coli can be found in many different habitats and has to be prepared to survive and grow under unfavorable conditions. Bacteria adaptation to different growth conditions requires an efficient control of gene expression. One of the primary forms of gene expression control is the competition between different sigma factors for the binding to the core RNA polymerase. d70 is the most abundant sigma factor and participates in the transcription of most E. coli genes. dS is the second one in importance and recognizes promoters of genes related to the general stress response. The rpoS gene, which encodes dS, is highly polymorphic and acquires mutations very often. The transition C®T at position 97 in the rpoS ORF results in a stop codon TAG (amber). Due to the presence of an alternative Shine-Dalgarno and a translation initiation codon at position 157 a truncated RpoS protein that is partially functional is translated. One of the stress situations that dS is activated is the starvation for inorganic phosphate. Phosphate limitation also triggers the activation of the PHO regulon, whose genes are predominantly transcribed by d70. In the present study, the effect of the truncated version of RpoS on the expression of d70 dependent genes (lacZ, phoA e pstS) and dS dependent genes (osmY e proU) was tested. Bacteria were also assayed for sensitivity to oxidative, osmotic and cold stress. The profile of partial activity described for the truncated RpoS could be observed in some cases, while in others the behavior of this allele resembled the rpoS null mutant. In parallel, an E. coli strain which suppresses the amber mutation in RpoS, resulting in the expression of a normal protein was also tested. A band correponding to the truncated RpoS could not be detected in immunoblots probably due to inefficient translation from the alternative Shine-Dalgarno. To improve the detection of RpoS, a highly immunogenic SPA tag was inserted in the C-terminal region of the protein

Characterization of Escherichia coli strains carrying different rpos alleles.

A bactéria Escherichia coli é encontrada em diversos habitats e deve estar preparada para sobreviver e crescer em condições desfavoráveis. A adaptação da bactéria a diferentes condições obriga-a a controlar a expressão de genes de forma eficiente. Uma das formas primárias de controle de expressão gênica é a competição entre os diversos fatores sigma pela ligação ao cerne da RNA polimerase. d70 é o fator sigma mais abundante e participa da transcrição da maioria dos genes de E. coli, enquanto que dS é o segundo em importância e reconhece promotores de genes relacionados à resposta geral ao estresse. O gene rpoS, que codifica para dS é altamente polimórfico, e adquiri mutações frequentemente. A mutação pontual C®T na posição 97 da ORF de rpoS, resulta em um códon de parada TAG (âmbar). Um Shine-Dalgarno alternativo e um códon de início de tradução na posição 157 dá início a uma proteína RpoS truncada, que é parcialmente funcional. Uma das situações de estresse na qual dS é ativado corresponde à privação de fosfato inorgânico. Porém, na limitação deste nutriente ocorre também a ativação do regulon PHO, cujos genes são predominantemente transcritos por d70. Neste trabalho, o efeito da versão truncada de RpoS sobre a expressão de genes dependentes de d70 (lacZ, phoA e pstS) e de genes dependentes de dS (osmY e proU) foi testado. Foram também realizados ensaios de estresse oxidativo, osmótico e pelo frio. O perfil de atividade parcial descrito para RpoSam pôde ser observado em alguns casos, porém em outros o comportamento deste alelo se assemelhou ao do mutante rpoS nulo. Paralelamente, foi testada também uma cepa de E. coli que carrega a mutação âmbar em rpoS, mas esta é suprimida, resultando na expressão de uma proteína RpoS normal. A proteína RpoS truncada não pôde ser visualizada em immunoblots, provavelmente porque esta é traduzida de forma pouco eficiente a partir do Shine-Dalgarno alternativo. Com o objetivo de incrementar a detecção de RpoS em ensaios de imuno-detecção, foi inserida por recombinação alélica uma etiqueta SPA altamente imunogênica na porção C-terminal da proteína.Escherichia coli can be found in many different habitats and has to be prepared to survive and grow under unfavorable conditions. Bacteria adaptation to different growth conditions requires an efficient control of gene expression. One of the primary forms of gene expression control is the competition between different sigma factors for the binding to the core RNA polymerase. d70 is the most abundant sigma factor and participates in the transcription of most E. coli genes. dS is the second one in importance and recognizes promoters of genes related to the general stress response. The rpoS gene, which encodes dS, is highly polymorphic and acquires mutations very often. The transition C®T at position 97 in the rpoS ORF results in a stop codon TAG (amber). Due to the presence of an alternative Shine-Dalgarno and a translation initiation codon at position 157 a truncated RpoS protein that is partially functional is translated. One of the stress situations that dS is activated is the starvation for inorganic phosphate. Phosphate limitation also triggers the activation of the PHO regulon, whose genes are predominantly transcribed by d70. In the present study, the effect of the truncated version of RpoS on the expression of d70 dependent genes (lacZ, phoA e pstS) and dS dependent genes (osmY e proU) was tested. Bacteria were also assayed for sensitivity to oxidative, osmotic and cold stress. The profile of partial activity described for the truncated RpoS could be observed in some cases, while in others the behavior of this allele resembled the rpoS null mutant. In parallel, an E. coli strain which suppresses the amber mutation in RpoS, resulting in the expression of a normal protein was also tested. A band correponding to the truncated RpoS could not be detected in immunoblots probably due to inefficient translation from the alternative Shine-Dalgarno. To improve the detection of RpoS, a highly immunogenic SPA tag was inserted in the C-terminal region of the protein

The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in <it>Escherichia coli</it>

Abstract Background Sigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor σS (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain. Results Nine ECORs had highly deleterious mutations in rpoS, 12 had RpoS protein up to 7-fold above that of the reference strain MG1655 and the remainder had comparable or lower levels. Strain variation was also evident in ppGpp accumulation under carbon starvation and spoT mutations were present in several low-ppGpp strains. Three relationships between RpoS and ppGpp levels were found: isolates with zero RpoS but various ppGpp levels, strains where RpoS levels were proportional to ppGpp and a third unexpected class in which RpoS was present but not proportional to ppGpp concentration. High-RpoS and high-ppGpp strains accumulated rpoS mutations under nutrient limitation, providing a source of polymorphisms. Conclusions The ppGpp and σS variance means that the expression of genes involved in translation, stress and other traits affected by ppGpp and/or RpoS are likely to be strain-specific and suggest that influential components of regulatory networks are frequently reset by microevolution. Different strains of E. coli have different relationships between ppGpp and RpoS levels and only some exhibit a proportionality between increasing ppGpp and RpoS levels as demonstrated for E. coli K-12.</p

The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in Escherichia coli

Abstract Background Sigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor σS (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain. Results Nine ECORs had highly deleterious mutations in rpoS, 12 had RpoS protein up to 7-fold above that of the reference strain MG1655 and the remainder had comparable or lower levels. Strain variation was also evident in ppGpp accumulation under carbon starvation and spoT mutations were present in several low-ppGpp strains. Three relationships between RpoS and ppGpp levels were found: isolates with zero RpoS but various ppGpp levels, strains where RpoS levels were proportional to ppGpp and a third unexpected class in which RpoS was present but not proportional to ppGpp concentration. High-RpoS and high-ppGpp strains accumulated rpoS mutations under nutrient limitation, providing a source of polymorphisms. Conclusions The ppGpp and σS variance means that the expression of genes involved in translation, stress and other traits affected by ppGpp and/or RpoS are likely to be strain-specific and suggest that influential components of regulatory networks are frequently reset by microevolution. Different strains of E. coli have different relationships between ppGpp and RpoS levels and only some exhibit a proportionality between increasing ppGpp and RpoS levels as demonstrated for E. coli K-12

Trans-splicing of mRNAs links gene transcription to translational control regulated by mTOR

Background: In phylogenetically diverse organisms, the 5′ ends of a subset of mRNAs are trans-spliced with a spliced leader (SL) RNA. The functions of SL trans-splicing, however, remain largely enigmatic. Results: We quantified translation genome-wide in the marine chordate, Oikopleura dioica, under inhibition of mTOR, a central growth regulator. Translation of trans-spliced TOP mRNAs was suppressed, consistent with a role of the SL sequence in nutrient-dependent translational control of growth-related mRNAs. Under crowded, nutrient-limiting conditions, O. dioica continued to filter-feed, but arrested growth until favorable conditions returned. Upon release from unfavorable conditions, initial recovery was independent of nutrient-responsive, trans-spliced genes, suggesting animal density sensing as a first trigger for resumption of development. Conclusion: Our results are consistent with a proposed role of trans-splicing in the coordinated translational down-regulation of nutrient-responsive genes under growth-limiting conditions