Transforming DNA Uptake Gene Orthologs Do Not Mediate Spontaneous Plasmid Transformation in
            <i>Escherichia coli</i>

Sun, Dongchang; Zhang, Xuewu; Wang, Lingyu; Prudhomme, Marc; Xie, Zhaoxiong; Martin, Bernard; Claverys, Jean‐Pierre

doi:10.1128/jb.01130-08

Cited by 30 publications

(43 citation statements)

References 23 publications

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“…Our further investigation revealed that plasmid transformation on plates is promoted by agar/agarose, a stimulation that is unrelated to divalent cations like Ca 2+ , Mg 2+ and Mn 2+ [10]. Interestingly, none of the DNA uptake gene homologs were found to be involved in mediating spontaneous plasmid transformation of E. coli [10]. The dose-response curve of transformation frequency as a function of DNA concentration showed that E. coli cells acquired plasmid DNA with a single hit kinetics, suggesting that plasmid DNA enters E. coli cells through a different route which allows double-stranded DNA (dsDNA) entry [10].…”

Section: Introductionmentioning

confidence: 77%

“…3) [10]. A recent report indicates that cell-to-cell transformation in E. coli may be regulated by a pheromone [28].…”

Section: Discussionmentioning

confidence: 99%

“…In Vibrio cholerae , RpoS regulates natural competence for transformation through a quorum sensing regulator HapR which controls the transcription of a conserved DNA uptake gene [26]. Because our previous study showed that DNA uptake gene orthologs ppdD , hofQ , gspD and ycaI , together with a highly conserved transforming ssDNA processing gene dprA / smf , were not required for spontaneous plasmid transformation of E. coli [10], mechanisms of transformation regulation by RpoS and cell density should be different in E. coli and V. cholerae . Considering that dsDNA enters E. coli cells during spontaneous plasmid transformation [10], we envision that RpoS may regulate the transcription of the gene(s) which belong(s) to a new set of DNA uptake genes encoding membrane/periplasmic proteins for the passage of dsDNA and/or DNA processing genes that are required for plasmid establishment in E. coli .…”

Section: Discussionmentioning

confidence: 99%

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RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

Zhang

Shi

et al. 2012

PLoS ONE

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Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes ( osmC , yqjC , ygiW and ugpC ) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoS K173E mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes.

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Section: Introductionmentioning

confidence: 77%

“…3) [10]. A recent report indicates that cell-to-cell transformation in E. coli may be regulated by a pheromone [28].…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

Zhang

Shi

et al. 2012

PLoS ONE

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“…Reannealing of complementary ssDNA fragments in the bacterial cytoplasm can theoretically occur (Saunders and Guild, 1981;Lorenz and Wackernagel, 1994;Domingues et al, 2012). Complementary DNA strands have been reported as dsDNA within the bacterial cell wall and within the transformed bacterial cell (Sun et al, 2009). …”

Section: (I) Exposure To Dnamentioning

confidence: 99%

Scientific Opinion on an application (EFSA-GMO-NL-2009-70) for the placing on the market of genetically modified drought tolerant maize MON 87460 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto

2012

EFSA Journal

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Maize MON 87460 was developed through Agrobacterium-mediated transformation and expresses the cold shock protein B (CspB) from Bacillus subtilis and neomycin phosphotransferase II (NPTII) from Escherichia coli to reduce yield loss under water-limited conditions. Maize MON 87460 contains a single copy of the cspB and nptII expression cassettes. Bioinformatic analysis of the flanking sequences and the open reading frames spanning the junctions created by the transformation did not raise safety issues. Comparative analyses established that, besides the expression of the CspB and NPTII proteins, some differences were observed in the composition of forage and grain produced from maize MON 87460 compared with its conventional counterpart, when grown under well-watered conditions. Given the magnitude of these changes and the characteristics of these endpoints, the EFSA GMO Panel concluded that the observed differences do not raise safety concerns for humans and animals. Under stressful conditions, maize MON 87460 can show enhanced agronomic performance characteristics and some differences in chemical composition in comparison with its conventional counterpart. Given the intended trait, the observed differences were not unexpected, and did indicate no safety concerns. The safety assessment identified no concerns regarding the potential toxicity and allergenicity of the CspB and NPTII proteins, or of maize MON 87460. Maize MON 87460 is as nutritious as any other maize and can be used in the same way. In delivering its Scientific Opinion, the EFSA GMO Panel considered: the application EFSA-GMO-NL-2009-70; additional information supplied by the applicant; scientific comments submitted by the Member States; and relevant scientific publications.The EFSA GMO Panel evaluated maize MON 87460 with reference to the intended uses and principles described in its risk assessment and monitoring guidelines. The scientific evaluation of the risk assessment included molecular characterisation of the inserted DNA and expression of the target proteins. An evaluation of the comparative analysis of composition and agronomic and phenotypic traits was undertaken, and the safety of the newly expressed proteins and the whole food/feed were evaluated with respect to potential toxicity, allergenicity and nutritional quality. An evaluation of environmental impacts and of the post-market environmental monitoring plan was undertaken.Maize MON 87460 was developed through Agrobacterium-mediated transformation and expresses the cold shock protein B (CspB) from Bacillus subtilis and neomycin phosphotransferase II (NPTII) from Tn5 of Escherichia coli. Maize MON 87460 was developed to provide reduced yield loss under conditions in which water is limited compared with conventional maize. The CspB protein is an RNA chaperone associated with enhanced abiotic stress tolerance in bacteria and plants, through its interaction with RNA secondary structures, limiting their misfolding and allowing cells to maintain cellular functions under various stress co...

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“…Our previous research showed that E. coli could develop natural competence and take up plasmid DNA on agar plates in the absence of either additional Ca 2ϩ or heat shock (8). Furthermore, we found that DNA uptake gene orthologs required for other naturally transformable species were not involved in this transformation system (9), which implied that a different molecular mechanism of competence development and DNA uptake was being used. As reported by our group and others, the general stress response regulator RpoS ( S ) was shown to mediate natural transformation (10), and transformation frequencies were apparently increased when rpoS was induced in the liquid culture stage but did not change significantly when rpoS was induced on solid medium (11).…”

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confidence: 99%

Positive Effect of Carbon Sources on Natural Transformation in Escherichia coli: Role of Low-Level Cyclic AMP (cAMP)-cAMP Receptor Protein in the Derepression of rpoS

et al. 2015

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Natural plasmid transformation of Escherichia coli is a complex process that occurs strictly on agar plates and requires the global stress response factor S . Here, we showed that additional carbon sources could significantly enhance the transformability of E. coli. Inactivation of phosphotransferase system genes (ptsH, ptsG, and crr) caused an increase in the transformation frequency, and the addition of cyclic AMP (cAMP) neutralized the promotional effect of carbon sources. This implies a negative role of cAMP in natural transformation. Further study showed that crp and cyaA mutations conferred a higher transformation frequency, suggesting that the cAMP-cAMP receptor protein (CRP) complex has an inhibitory effect on transformation. Moreover, we observed that rpoS is negatively regulated by cAMP-CRP in early log phase and that both crp and cyaA mutants show no transformation superiority when rpoS is knocked out. Therefore, it can be concluded that both the crp and cyaA mutations derepress rpoS expression in early log phase, whereby they aid in the promotion of natural transformation ability. We also showed that the accumulation of RpoS during early log phase can account for the enhanced transformation aroused by additional carbon sources. Our results thus demonstrated that the presence of additional carbon sources promotes competence development and natural transformation by reducing cAMP-CRP and, thus, derepressing rpoS expression during log phase. This finding could contribute to a better understanding of the relationship between nutrition state and competence, as well as the mechanism of natural plasmid transformation in E. coli. IMPORTANCEEscherichia coli, which is not usually considered to be naturally transformable, was found to spontaneously take up plasmid DNA on agar plates. Researching the mechanism of natural transformation is important for understanding the role of transformation in evolution, as well as in the transfer of pathogenicity and antibiotic resistance genes. In this work, we found that carbon sources significantly improve transformation by decreasing cAMP. Then, the low level of cAMP-CRP derepresses the general stress response regulator RpoS via a biphasic regulatory pattern, thereby contributing to transformation. Thus, we demonstrate the mechanism by which carbon sources affect natural transformation, which is important for revealing information about the interplay between nutrition state and competence development in E. coli. Horizontal gene transfer (HGT) is the transfer of genes between distantly related organisms. It is widely recognized that HGT contributes significantly to the evolution of bacterial genomes and the adaptation of bacteria to new environments (1, 2). In bacteria, the physical process of DNA transfer is accomplished by transduction, conjugation, and transformation. Natural transformation is characterized by the spontaneous uptake of free DNA from the environment by a competent cell, which then integrates said DNA into its chromosome or stabilizes the DNA extr...

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Transforming DNA Uptake Gene Orthologs Do Not Mediate Spontaneous Plasmid Transformation in Escherichia coli

Cited by 30 publications

References 23 publications

RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

Scientific Opinion on an application (EFSA-GMO-NL-2009-70) for the placing on the market of genetically modified drought tolerant maize MON 87460 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto

Positive Effect of Carbon Sources on Natural Transformation in Escherichia coli: Role of Low-Level Cyclic AMP (cAMP)-cAMP Receptor Protein in the Derepression of rpoS

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