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Common requirement for the relaxosome of plasmid R1 in multiple activities of the conjugative type IV secretion system.

ABSTRACT: Macromolecular transport by bacterial type IV secretion systems involves regulated uptake of (nucleo)protein complexes by the cell envelope-spanning transport channel. A coupling protein receptor is believed to recognize the specific proteins destined for transfer, but the steps initiating their translocation remain unknown. Here, we investigate the contribution of a complex of transfer initiation proteins, the relaxosome, of plasmid R1 to translocation of competing transferable substrates from mobilizable plasmids ColE1 and CloDF13 or the bacteriophage R17. We found that not only does the R1 translocation machinery engage the R1 relaxosome during conjugative self-transfer and during infection by R17 phage but it is also activated by its cognate relaxosome to mediate the export of an alternative plasmid. Transporter activity was optimized by the R1 relaxosome even when this complex itself could not be transferred, i.e., when the N-terminal activation domain (amino acids 1 to 992 [N1-992]) of TraI was present without the C-terminal conjugative helicase domain. We propose that the functional dependence of the transfer machinery on the R1 relaxosome for initiating translocation ensures that dissemination of heterologous plasmids does not occur at the expense of self-transfer.

SUBMITTER: Lang S

PROVIDER: S-EPMC4010989 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Common requirement for the relaxosome of plasmid R1 in multiple activities of the conjugative type IV secretion system.

Lang Silvia S Gruber Christian J CJ Raffl Sandra S Reisner Andreas A Zechner Ellen L EL

Journal of bacteriology 20140328 11

Macromolecular transport by bacterial type IV secretion systems involves regulated uptake of (nucleo)protein complexes by the cell envelope-spanning transport channel. A coupling protein receptor is believed to recognize the specific proteins destined for transfer, but the steps initiating their translocation remain unknown. Here, we investigate the contribution of a complex of transfer initiation proteins, the relaxosome, of plasmid R1 to translocation of competing transferable substrates from ...[more]

PMID: 24682328

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Project description:UnlabelledConjugative plasmids have been identified in a wide variety of different bacteria, ranging from proteobacteria to firmicutes, and conjugation is one of the most efficient routes for horizontal gene transfer. The most widespread mechanism of plasmid conjugation relies on different variants of the type IV secretion pathway. Here, we describe the identification of a novel type of conjugative plasmid that seems to be unique for mycobacteria. Interestingly, while this plasmid is efficiently exchanged between different species of slow-growing mycobacteria, including Mycobacterium tuberculosis, it could not be transferred to any of the fast-growing mycobacteria tested. Genetic analysis of the conjugative plasmid showed the presence of a locus containing homologues of three type IV secretion system components and a relaxase. In addition, a new type VII secretion locus was present. Using transposon insertion mutagenesis, we show that in fact both these secretion systems are essential for conjugation, indicating that this plasmid represents a new class of conjugative plasmids requiring two secretion machineries. This plasmid could form a useful new tool to exchange or introduce DNA in slow-growing mycobacteria.ImportanceConjugative plasmids play an important role in horizontal gene transfer between different bacteria and, as such, in their adaptation and evolution. This effect is most obvious in the spread of antibiotic resistance genes. Thus far, conjugation of natural plasmids has been described only rarely for mycobacterial species. In fact, it is generally accepted that M. tuberculosis does not show any recent sign of horizontal gene transfer. In this study, we describe the identification of a new widespread conjugative plasmid that can also be efficiently transferred to M. tuberculosis. This plasmid therefore poses both a threat and an opportunity. The threat is that, through the acquisition of antibiotic resistance markers, this plasmid could start a rapid spread of antibiotic resistance genes between pathogenic mycobacteria. The opportunity is that we could use this plasmid to generate new tools for the efficient introduction of foreign DNA in slow-growing mycobacteria.

Project description:Differential gene expression in biofilm cells suggests that adding the derepressed conjugative plasmid R1drd19 increases biofilm formation by affecting genes related to envelope stress (rseA and cpxAR), biofilm formation (bssR and cstA), energy production (glpDFK), acid resistance (gadABCEX and hdeABD), and cell motility (csgBEFG, yehCD, yadC, and yfcV); genes encoding outer membrane proteins (ompACF), phage shock proteins (pspABCDE), and cold shock proteins (cspACDEG); and phage-related genes. To investigate the link between the identified genes and biofilm formation upon the addition of R1drd19, 40 isogenic mutants were classified according to their different biofilm formation phenotypes. Cells with class I mutations (those in rseA, bssR, cpxA, and ompA) exhibited no difference from the wild-type strain in biofilm formation and no increase in biofilm formation upon the addition of R1drd19. Cells with class II mutations (those in gatC, yagI, ompC, cspA, pspD, pspB, ymgB, gadC, pspC, ymgA, slp, cpxP, cpxR, cstA, rseC, ompF, and yqjD) displayed increased biofilm formation compared to the wild-type strain but decreased biofilm formation upon the addition of R1drd19. Class III mutants showed increased biofilm formation compared to the wild-type strain and increased biofilm formation upon the addition of R1drd19. Cells with class IV mutations displayed increased biofilm formation compared to the wild-type strain but little difference upon the addition of R1drd19, and class V mutants exhibited no difference from the wild-type strain but increased biofilm formation upon the addition of R1drd19. Therefore, proteins encoded by the genes corresponding to the class I mutant phenotype are involved in R1drd19-promoted biofilm formation, primarily through their impact on cell motility. We hypothesize that the pili formed upon the addition of the conjugative plasmid disrupt the membrane (induce ompA) and activate the two-component system CpxAR as well as the other envelope stress response system, RseA-sigma(E), both of which, along with BssR, play a key role in bacterial biofilm formation.

Project description:The pathogenic pVA1-type plasmids that carry pirAB toxin genes are the genetic basis for Vibrio to cause acute hepatopancreatic necrosis disease (AHPND), a lethal shrimp disease posing an urgent threat to shrimp aquaculture. Emerging evidence also demonstrate the rapid spread of pVA1-type plasmids across Vibrio species. The pVA1-type plasmids have been predicted to encode a self-encoded type IV secretion system (T4SS). Here, phylogenetic analysis indicated that the T4SS is a novel member of Trb-type. We further confirmed that the T4SS was able to mediate the conjugation of pVA1-type plasmids. A trbE gene encoding an ATPase and a traG gene annotated as a type IV coupling protein (T4CP) were characterized as key components of the T4SS. Deleting either of these 2 genes abolished the conjugative transfer of a pVA1-type plasmid from AHPND-causing Vibrio parahaemolyticus to Vibrio campbellii, which was restored by complementation of the corresponding gene. Moreover, we found that bacterial density, temperature, and nutrient levels are factors that can regulate conjugation efficiency. In conclusion, we proved that the conjugation of pVA1-type plasmids across Vibrio spp. is mediated by a novel T4SS and regulated by environmental factors. IMPORTANCE AHPND is a global shrimp bacteriosis and was listed as a notifiable disease by the World Organization for Animal Health (WOAH) in 2016, causing losses of more than USD 7 billion each year. Several Vibrio species such as V. parahaemolyticus, V. harveyi, V. campbellii, and V. owensii harboring the virulence plasmid (designated as the pVA1-type plasmid) can cause AHPND. The increasing number of Vibrio species makes prevention and control more difficult, threatening the sustainable development of the aquaculture industry. In this study, we found that the horizontal transfer of pVA1-type plasmid is mediated by a novel type IV secretion system (T4SS). Our study explained the formation mechanism of pathogen diversity in AHPND. Moreover, bacterial density, temperature, and nutrient levels can regulate horizontal efficiency. We explore new ideas for controlling the spread of virulence plasmid and form the basis of management strategies leading to the prevention and control of AHPND.

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Common requirement for the relaxosome of plasmid R1 in multiple activities of the conjugative type IV secretion system.

Publications

Common requirement for the relaxosome of plasmid R1 in multiple activities of the conjugative type IV secretion system.

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