Inactivation of the dimeric RappLS20 anti-repressor of the conjugation operon is mediated by peptide-induced tetramerization

Bernardo, Nerea; Miguel-Arribas, Andrés; Singh, Praveen; Luque‐Ortega, Juan Román; Alfonso, Carlos; Malfois, Marc; Meijer, Wilfried J. J.; Boer, Roeland

doi:10.1093/nar/gkaa540

Cited by 4 publications

(15 citation statements)

References 50 publications

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“…disruption of these complexes and the appearance of elution and SAXS patterns that were similar to those of Rco pLS20 alone and Rap pLS20 -Phr* pLS20 complexes (i.e., Rap pLS20 tetramers) (Crespo et al, 2020).…”

Section: The Activation State Of the Conjugation Genes Is Ultimately mentioning

confidence: 75%

“…Instead, Rap pLS20 seems to exert its antirepressor activity by binding directly to Rco pLS20 , as bacterial two-hybrid analysis showed that Rap pLS20 and Rco pLS20 interact in vivo ( Singh et al, 2020 ). In vitro evidence that Rap pLS20 interacts directly with Rco pLS20 has also been obtained by SAXS and analytical size exclusion chromatography ( Crespo et al, 2020 ). Furthermore, AUC analyses provided conclusive evidence that Rap pLS20 forms predominantly dimers in solution.…”

Section: Rap Pls20 Activates Conjugation By Detachmentioning

confidence: 96%

“…Gel retardation using a mixture of the small and large DNA fragment resulted in the appearance of an additional retarded species composed of a small and a large DNA fragment ( Singh et al, 2020 ). Furthermore, SAXS analysis showed that Rco pLS20 forms complexes of increased size at concentrations above 200 μM, indicating that Rco pLS20 tetramers can interact with each other ( Crespo et al, 2020 ), which would be expected if DNA binding occurs via the second mode. The combined evidences strongly indicate that efficient repression of the P c promoter and simultaneous activation of the P r promoter by Rco pLS20 is achieved through DNA looping as a consequence of interaction between the Rco pLS20 tetramers bound to operators O I and O II .…”

Section: Regulation Of P C and P Rmentioning

confidence: 99%

“…Based on the features of the known RRNPP proteins, one possibility of how Rap pLS20 might activate the pLS20 conjugation genes is by competing with Rco pLS20 for binding to DNA near the P c promoter. However, both gel retardation and analytical ultracentrifugation (AUC) analyses showed that Rap pLS20 has no DNA binding activity, and the NTD of Rap pLS20 does not adopt a HTH configuration ( Crespo et al, 2020 ; Singh et al, 2020 ). Instead, Rap pLS20 seems to exert its antirepressor activity by binding directly to Rco pLS20 , as bacterial two-hybrid analysis showed that Rap pLS20 and Rco pLS20 interact in vivo ( Singh et al, 2020 ).…”

Section: Rap Pls20 Activates Conjugation By Detachmentioning

confidence: 99%

“…Phr* pLS20 binds directly to Rap pLS20 (see below) and the dissociation constant, K d , for Phr* pLS20 was determined to be 2.1 and 7.4 μM by ultracentrifugation and fluorescence anisotropy approaches, respectively ( Crespo et al, 2020 ; Singh et al, 2020 ). Although AUC analysis showed that Rap pLS20 forms predominantly dimers in solution ( Crespo et al, 2020 , see also above), it seems to have an intrinsic ability to form tetramers at higher concentrations, as 1.1% of Rap pLS20 was detected as tetramers in solution at 4.5 μM, and this percentage increased to 3.6% at 25 μM. Interestingly, AUC analysis showed that in the presence of Phr* pLS20 , Rap pLS20 forms almost exclusively tetramers in solution ( Crespo et al, 2020 ).…”

Section: The Activation State Of the Conjugation Genes Is Ultimately mentioning

confidence: 99%

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Multiple Layered Control of the Conjugation Process of the Bacillus subtilis Plasmid pLS20

Meijer

Boer

Ares

et al. 2021

Front. Mol. Biosci.

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Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter Pc located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum-signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the Pc promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed.

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Section: The Activation State Of the Conjugation Genes Is Ultimately mentioning

confidence: 75%

Section: Rap Pls20 Activates Conjugation By Detachmentioning

confidence: 96%

Section: Regulation Of P C and P Rmentioning

confidence: 99%

Section: Rap Pls20 Activates Conjugation By Detachmentioning

confidence: 99%

Section: The Activation State Of the Conjugation Genes Is Ultimately mentioning

confidence: 99%

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Multiple Layered Control of the Conjugation Process of the Bacillus subtilis Plasmid pLS20

Meijer

Boer

Ares

et al. 2021

Front. Mol. Biosci.

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Reversible regulation of conjugation of Bacillus subtilis plasmid pLS20 by the quorum sensing peptide responsive anti-repressor RappLS20

Singh

Serrano

Ramachandran

et al. 2020

Nucleic Acids Research

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Quorum sensing plays crucial roles in bacterial communication including in the process of conjugation, which has large economical and health-related impacts by spreading antibiotic resistance. The conjugative Bacillus subtilis plasmid pLS20 uses quorum sensing to determine when to activate the conjugation genes. The main conjugation promoter, Pc, is by default repressed by a regulator RcopLS20 involving DNA looping. A plasmid-encoded signalling peptide, Phr*pLS20, inactivates the anti-repressor of RcopLS20, named RappLS20, which belongs to the large group of RRNPP family of regulatory proteins. Here we show that DNA looping occurs through interactions between two RcopLS20 tetramers, each bound to an operator site. We determined the relative promoter strengths for all the promoters involved in synthesizing the regulatory proteins of the conjugation genes, and constructed an in vivo system uncoupling these regulatory genes to show that RappLS20 is sufficient for activating conjugation in vivo. We also show that RappLS20 actively detaches RcopLS20 from DNA by preferentially acting on the RcopLS20 molecules involved in DNA looping, resulting in sequestration but not inactivation of RcopLS20. Finally, results presented here in combination with our previous results show that activation of conjugation inhibits competence and competence development inhibits conjugation, indicating that both processes are mutually exclusive.

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pLS20 is the archetype of a new family of conjugative plasmids harboured byBacillusspecies

Val-Calvo

Miguel-Arribas

Abia

et al. 2021

NAR Genomics and Bioinformatics

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Conjugation plays important roles in genome plasticity, adaptation and evolution but is also the major horizontal gene-transfer route responsible for spreading toxin, virulence and antibiotic resistance genes. A better understanding of the conjugation process is required for developing drugs and strategies to impede the conjugation-mediated spread of these genes. So far, only a limited number of conjugative elements have been studied. For most of them, it is not known whether they represent a group of conjugative elements, nor about their distribution patterns. Here we show that pLS20 from the Gram-positive bacterium Bacillus subtilis is the prototype conjugative plasmid of a family of at least 35 members that can be divided into four clades, and which are harboured by different Bacillus species found in different global locations and environmental niches. Analyses of their phylogenetic relationship and their conjugation operons have expanded our understanding of a family of conjugative plasmids of Gram-positive origin.

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Inactivation of the dimeric RappLS20 anti-repressor of the conjugation operon is mediated by peptide-induced tetramerization

Cited by 4 publications

References 50 publications

Multiple Layered Control of the Conjugation Process of the Bacillus subtilis Plasmid pLS20

Multiple Layered Control of the Conjugation Process of the Bacillus subtilis Plasmid pLS20

Reversible regulation of conjugation of Bacillus subtilis plasmid pLS20 by the quorum sensing peptide responsive anti-repressor RappLS20

pLS20 is the archetype of a new family of conjugative plasmids harboured byBacillusspecies

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