Conjugative plasmid transfer in gram-positive bacteria.
ABSTRACT: Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.
Project description:Conjugative plasmids are the main players in horizontal gene transfer in Gram-negative bacteria. DNA transfer tools constructed on the basis of such plasmids enable gene manipulation even in strains of clinical or environmental origin, which are often difficult to work with. The conjugation system of the IncM plasmid pCTX-M3 isolated from a clinical strain of Citrobacter freundii has been shown to enable efficient mobilization of oriT pCTX-M3-bearing plasmids into a broad range of hosts comprising Alpha-, Beta-, and Gammaproteobacteria We constructed a helper plasmid, pMOBS, mediating such mobilization with an efficiency up to 1,000-fold higher than that achieved with native pCTX-M3. We also constructed Escherichia coli donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator (orf35) of the pCTX-M3 tra genes, and S25 and S26, devoid of two putative regulators (orf35 and orf36) of the pCTX-M3 tra genes. Strains S14 and S15 and strains S25 and S26 are, respectively, up to 100 and 1,000 times more efficient in mobilization than pCTX-M3. Moreover, they also enable plasmid mobilization into the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis Additionally, the constructed E. coli strains carried no antibiotic resistance genes that are present in pCTX-M3 to facilitate manipulations with antibiotic-resistant recipient strains, such as those of clinical origin. To demonstrate possible application of the constructed tool, an antibacterial conjugation-based system was designed. Strain S26 was used for introduction of a mobilizable plasmid coding for a toxin, resulting in the elimination of over 90% of recipient E. coli cells.IMPORTANCE The conjugation of donor and recipient bacterial cells resulting in conjugative transfer of mobilizable plasmids is the preferred method enabling the introduction of DNA into strains for which other transfer methods are difficult to establish (e.g., clinical strains). We have constructed E. coli strains carrying the conjugation system of the IncM plasmid pCTX-M3 integrated into the chromosome. To increase the mobilization efficiency up to 1,000-fold, two putative regulators of this system, orf35 and orf36, were disabled. The constructed strains broaden the repertoire of tools for the introduction of DNA into the Gram-negative Alpha-, Beta-, and Gammaproteobacteria, as well as into Gram-positive bacteria such as Bacillus subtilis and Lactococcus lactis The antibacterial procedure based on conjugation with the use of the orf35- and orf36-deficient strain lowered the recipient cell number by over 90% owing to the mobilizable plasmid-encoded toxin.
Project description:During conjugation a genetic element is transferred from a bacterial donor to a recipient cell via a connecting channel. It is the major route responsible for the spread of antibiotic resistance. Conjugative elements can contain exclusion system(s) that inhibit its transfer to a cell already harboring the element. Our limited knowledge on exclusion systems is mainly based on plasmids of Gram-negative bacteria. Here we studied the conjugative plasmid pLS20 of the Gram-positive <i>Bacillus subtilis</i>. We demonstrate that pLS20 contains an exclusion system and identified the single gene responsible for exclusion, named <i>ses<sub>pLS20</sub></i> , which is embedded in the conjugation operon. Ses<sub>pLS20</sub> is the founding member of a novel family of surface exclusion proteins encoded by conjugative elements of Gram-positive origin. We show that the extent of surface exclusion correlates with the level of <i>ses<sub>pLS20</sub></i> expression, and that <i>ses<sub>pLS20</sub></i> is expressed at basal low-levels in all donor cells but becomes highly expressed in conjugating cells. Accordingly, the transfer of pLS20 from a conjugation-primed donor cell to an un-primed or conjugation-primed donor is inhibited moderately and very efficiently, respectively. The consequences of this differential regulation, which appears to be a conserved feature of surface exclusion systems of Gram-positive and Gram-negative origin, are discussed.
Project description:Bacterial conjugation presents the most important means to spread antibiotic resistance and virulence factors among closely and distantly related bacteria. Conjugative plasmids are the mobile genetic elements mainly responsible for this task. All the genetic information required for the horizontal transmission is encoded on the conjugative plasmids themselves. Two distinct concepts for horizontal plasmid transfer in Gram-positive bacteria exist, the most prominent one transports single stranded plasmid DNA via a multi-protein complex, termed type IV secretion system, across the Gram-positive cell envelope. Type IV secretion systems have been found in virtually all unicellular Gram-positive bacteria, whereas multicellular Streptomycetes seem to have developed a specialized system more closely related to the machinery involved in bacterial cell division and sporulation, which transports double stranded DNA from donor to recipient cells. This review intends to summarize the state of the art of prototype systems belonging to the two distinct concepts; it focuses on protein key players identified so far and gives future directions for research in this emerging field of promiscuous interbacterial transport.
Project description:Conjugation, the process by which a DNA element is transferred from a donor to a recipient cell, is the main horizontal gene transfer route responsible for the spread of antibiotic resistance and virulence genes. Contact between a donor and a recipient cell is a prerequisite for conjugation, because conjugative DNA is transferred into the recipient via a channel connecting the two cells. Conjugative elements encode proteins dedicated to facilitating the recognition and attachment to recipient cells, also known as mating pair formation. A subgroup of the conjugative elements is able to mediate efficient conjugation during planktonic growth, and mechanisms facilitating mating pair formation will be particularly important in these cases. Conjugative elements of Gram-negative bacteria encode conjugative pili, also known as sex pili, some of which are retractile. Far less is known about mechanisms that promote mating pair formation in Gram-positive bacteria. The conjugative plasmid pLS20 of the Gram-positive bacterium <i>Bacillus subtilis</i> allows efficient conjugation in liquid medium. Here, we report the identification of an adhesin gene in the pLS20 conjugation operon. The N-terminal region of the adhesin contains a class II type thioester domain (TED) that is essential for efficient conjugation, particularly in liquid medium. We show that TED-containing adhesins are widely conserved in Gram-positive bacteria, including pathogens where they often play crucial roles in pathogenesis. Our study is the first to demonstrate the involvement of a class II type TED-containing adhesin in conjugation.<b>IMPORTANCE</b> Bacterial resistance to antibiotics has become a serious health care problem. The spread of antibiotic resistance genes between bacteria of the same or different species is often mediated by a process named conjugation, where a donor cell transfers DNA to a recipient cell through a connecting channel. The first step in conjugation is recognition and attachment of the donor to a recipient cell. Little is known about this first step, particularly in Gram-positive bacteria. Here, we show that the conjugative plasmid pLS20 of <i>Bacillus subtilis</i> encodes an adhesin protein that is essential for effective conjugation. This adhesin protein has a structural organization similar to adhesins produced by other Gram-positive bacteria, including major pathogens, where the adhesins serve in attachment to host tissues during colonization and infection. Our findings may thus also open novel avenues to design drugs that inhibit the spread of antibiotic resistance by blocking the first recipient-attachment step in conjugation.
Project description:Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.
Project description:The increase of infections caused by multidrug-resistant bacteria, together with the loss of effectiveness of currently available antibiotics, represents one of the most serious threats to public health worldwide. The loss of human lives and the economic costs associated to the problem of the dissemination of antibiotic resistance require immediate action. Bacteria, known by their great genetic plasticity, are capable not only of mutating their genes to adapt to disturbances and environmental changes but also of acquiring new genes that allow them to survive in hostile environments, such as in the presence of antibiotics. One of the major mechanisms responsible for the horizontal acquisition of new genes (e.g., antibiotic resistance genes) is bacterial conjugation, a process mediated by mobile genetic elements such as conjugative plasmids and integrative conjugative elements. Conjugative plasmids harboring antibiotic resistance genes can be transferred from a donor to a recipient bacterium in a process that requires physical contact. After conjugation, the recipient bacterium not only harbors the antibiotic resistance genes but it can also transfer the acquired plasmid to other bacteria, thus contributing to the spread of antibiotic resistance. Conjugative plasmids have genes that encode all the proteins necessary for the conjugation to take place, such as the type IV coupling proteins (T4CPs) present in all conjugative plasmids. Type VI coupling proteins constitute a heterogeneous family of hexameric ATPases that use energy from the ATP hydrolysis for plasmid transfer. Taking into account their essential role in bacterial conjugation, T4CPs are attractive targets for the inhibition of bacterial conjugation and, concomitantly, the limitation of antibiotic resistance dissemination. This review aims to compile present knowledge on T4CPs as a starting point for delving into their molecular structure and functioning in future studies. Likewise, the scientific literature on bacterial conjugation inhibitors has been reviewed here, in an attempt to elucidate the possibility of designing T4CP-inhibitors as a potential solution to the dissemination of multidrug-resistant bacteria.
Project description:The emergence of pathogenic bacteria resistant to multiple antibiotics is a serious worldwide public health concern. Whenever antibiotics are applied, the genes encoding for antibiotic resistance are selected for within bacterial populations. This has led to the prevalence of conjugative plasmids that carry resistance genes and can transfer themselves between diverse bacterial groups. In this study, we investigated whether it is feasible to attempt to prevent the spread of antibiotic resistances with a lytic bacteriophage, which can replicate in a wide range of gram-negative bacteria harbouring conjugative drug resistance-conferring plasmids. The counter-selection against the plasmid was shown to be effective, reducing the frequency of multidrug-resistant bacteria that formed via horizontal transfer by several orders of magnitude. This was true also in the presence of an antibiotic against which the plasmid provided resistance. Majority of the multiresistant bacteria subjected to phage selection also lost their conjugation capability. Overall this study suggests that, while we are obligated to maintain the selection for the spread of the drug resistances, the 'fight evolution with evolution' approach could help us even out the outcome to our favour.
Project description:Abstract 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.
Project description:BACKGROUND: The frequency of transfer of genes encoding resistance to antimicrobial agents was determined by conjugation in ESBL-producing and/or fluoroquinolone or aminoglycoside resistant Enterobacteriaceae clinical isolates at a tertiary care center in Lebanon. In addition, the role of tra genes encoding transferases in mediating conjugation was assessed. METHODS: Conjugation experiments were done on 53 ESBL-producing and/or fluoroquinolone resistant E. coli and K. pneumoniae and ESBL-producing S. sonnei isolates. Antimicrobial susceptibility testing on parent and transconjugant isolates, and PCR amplifications on plasmid extracts of the resistance-encoding genes: blaCTX-M-15 with the ISEcp1 insertion sequence, the aac(6')-lb-cr and qnrS genes, as well as tra encoding transferases genes were done. Random amplified polymorphic DNA (RAPD) analysis was performed to demonstrate whether conjugative isolates are clonal and whether they are linked epidemiologically to a particular source. RESULTS: Antimicrobial susceptibility testing on transconjugants revealed that 26 out of 53 (49%) ESBL-producing Enterobacteriaceae were able to transfer antimicrobial resistance to the recipients. Transfer of high-level resistance to the transconjugants encoded by the blaCTX-M-15 gene downstream the ISEcp1 insertion sequence against 3rd generation cephalosporins, and of low-level resistance against ciprofloxacin, and variable levels of resistance against aminoglycosides encoded by aac(6')-lb-cr gene, were observed in transconjugants. tra encoding transferase genes were detected exclusively in conjugative isolates. CONCLUSION: In conclusion, the frequency of transfer of antimicrobial resistance in non clonal Enterobacteriaceae at the tertiary care center by conjugation was 49%. Conjugation occurred in isolates expressing the tra encoding transferase genes. Multiple conjugative strains harboring the plasmid encoded antimicrobial resistant genes were circulating in the medical center. Molecular epidemiology analysis showed that conjugative isolates are neither clonal nor linked to a particular site and transfer of antimicrobial resistance is by horizontal transfer of plasmids.
Project description:Type IV Secretion Systems (T4SSs) are membrane-spanning multiprotein complexes dedicated to protein secretion or conjugative DNA transport (conjugation systems) in bacteria. The prototype and best-characterized T4SS is that of the Gram-negative soil bacterium Agrobacterium tumefaciens. For Gram-positive bacteria, only conjugative T4SSs have been characterized in some biochemical, structural, and mechanistic details. These conjugation systems are predominantly encoded by self-transmissible plasmids but are also increasingly detected on integrative and conjugative elements (ICEs) and transposons. Here, we report regulatory details of conjugation systems from Enterococcus model plasmids pIP501 and pCF10, Bacillus plasmid pLS1, Clostridium plasmid pCW3, and staphylococcal plasmid pSK41. In addition, regulation of conjugative processes of ICEs (ICEBs1, ICESt1, ICESt3) by master regulators belonging to diverse repressor families will be discussed. A special focus of this review lies on the comparison of regulatory mechanisms executed by proteins belonging to the RRNPP family. These regulators share a common fold and govern several essential bacterial processes, including conjugative transfer.