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Identification of a novel conjugative plasmid in mycobacteria that requires both type IV and type VII secretion.

ABSTRACT: UNLABELLED:Conjugative 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. IMPORTANCE:Conjugative 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.

SUBMITTER: Ummels R

PROVIDER: S-EPMC4173767 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Identification of a novel conjugative plasmid in mycobacteria that requires both type IV and type VII secretion.

Ummels Roy R Abdallah Abdallah M AM Kuiper Vincent V Aâjoud Anouar A Sparrius Marion M Naeem Raeece R Spaink Herman P HP van Soolingen Dick D Pain Arnab A Bitter Wilbert W

mBio 20140923 5

<h4>Unlabelled</h4>Conjugative 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 ef ...[more]

PMID: 25249284

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Project description:Tuberculosis, one of the world's most severe infectious diseases, is caused by Mycobacterium tuberculosis A major weapon of this pathogen is a unique cell wall that protects the pathogen from eradication by the immune system. Mycobacteria have specialized secretion systems, e.g., type VII secretion or ESX systems, to transport substrates across this cell wall. The largest group of proteins that are secreted by these ESX systems are the PE proteins. Previously, it was shown that the N-terminal PE domain of about 100 amino acids is required for secretion. Here, we describe the identification of an aspartic protease, designated PecA, that removes (part of) this PE domain at the cell surface. Nearly all of the observed PE_PGRS proteins are processed by PecA. Interestingly, the protease itself is also a secreted PE protein and subject to self-cleavage. Furthermore, a defect in surface processing has no effect on the activity of the PE lipase protein LipY but does seem to affect the functioning of other virulence factors, as a pecA mutant strain of Mycobacterium marinum shows moderate attenuation in zebrafish larvae. In conclusion, our results reveal the presence of a functional aspartic acid protease in M. marinum that cleaves LipY, itself as well as other members of the PE_PGRS family. Finally, mutants lacking PecA show growth attenuation in vivo, suggesting that PecA plays a role during infection.IMPORTANCE Aspartic proteases are common in eukaryotes and retroviruses but are relatively rare among bacteria (N. D. Rawlings and A. Bateman, BMC Genomics 10:437, 2009, https://doi.org/10.1186/1471-2164-10-437). In contrast to eukaryotic aspartic proteases, bacterial aspartic proteases are generally located in the cytoplasm. We have identified a surface-associated mycobacterial aspartic protease, PecA, which cleaves itself and many other type VII secretion substrates of the PE_PGRS family. PecA is present in most pathogenic mycobacterial species, including M. tuberculosis In addition, pathogenicity of M. marinum is reduced in the ?pecA mutant, indicating that PecA contributes to virulence.

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.

Project description:BackgroundThe genome of Mycobacterium tuberculosis contains five copies of the ESX gene cluster, each encoding a dedicated protein secretion system. These ESX secretion systems have been defined as a novel Type VII secretion machinery, responsible for the secretion of proteins across the characteristic outer mycomembrane of the mycobacteria. Some of these secretion systems are involved in virulence and survival in M. tuberculosis; however they are also present in other non-pathogenic mycobacteria, and have been identified in some non-mycobacterial actinomycetes. Three components of the ESX gene cluster have also been found clustered in some gram positive monoderm organisms and are predicted to have preceded the ESX gene cluster.ResultsThis study used in silico and phylogenetic analyses to describe the evolution of the ESX gene cluster from the WXG-FtsK cluster of monoderm bacteria to the five ESX clusters present in M. tuberculosis and other slow-growing mycobacteria. The ancestral gene cluster, ESX-4, was identified in several nonmycomembrane producing actinobacteria as well as the mycomembrane-containing Corynebacteriales in which the ESX cluster began to evolve and diversify. A novel ESX gene cluster, ESX-4EVOL, was identified in some non-mycobacterial actinomycetes and M. abscessus subsp. bolletii. ESX-4EVOL contains all of the conserved components of the ESX gene cluster and appears to be a precursor of the mycobacterial ESX duplications. Between two and seven ESX gene clusters were identified in each mycobacterial species, with ESX-2 and ESX-5 specifically associated with the slow growers. The order of ESX duplication in the mycobacteria is redefined as ESX-4, ESX-3, ESX-1 and then ESX-2 and ESX-5. Plasmid-encoded precursor ESX gene clusters were identified for each of the genomic ESX-3, -1, -2 and -5 gene clusters, suggesting a novel plasmid-mediated mechanism of ESX duplication and evolution.ConclusionsThe influence of the various ESX gene clusters on vital biological and virulence-related functions has clearly influenced the diversification and success of the various mycobacterial species, and their evolution from the non-pathogenic fast-growing saprophytic to the slow-growing pathogenic organisms.

Dataset Information

Identification of a novel conjugative plasmid in mycobacteria that requires both type IV and type VII secretion.

Publications

Identification of a novel conjugative plasmid in mycobacteria that requires both type IV and type VII secretion.

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