Transfer systems of K88 and K99 plasmids

The conjugation systems of three K88-mobilizing plasmids were characterized for the morphology of their pili and type of mating system (surface only or surface + liquid). pREI had a typical IncI1 transfer system with both thick and thin pili. pVIDO determined aggregating thick flexible pili and pPLS nonaggregating thick flexible pili. All three transferred equally well in broth and on plates. pPLS alone was naturally transfer-depressed. pREI and pVIDO were tested for K88 mobilization efficiency, which was greater from their wild-type host strains to Escherichia coli K-12 than between E. coli K-12 strains. The K99 conjugative plasmid from strain B41 was repressed for transfer and determined thick flexible pili that were receptors for the filamentous phage fd.     

Interplay between the cell envelope and mobile genetic elements shapes gene flow in populations of the nosocomial pathogen Klebsiella pneumoniae

Mobile genetic elements (MGEs) drive genetic transfers between bacteria using mechanisms that require a physical interaction with the cellular envelope. In the high-priority multidrug-resistant nosocomial pathogens (ESKAPE), the first point of contact between the cell and virions or conjugative pili is the capsule. While the capsule can be a barrier to MGEs, it also evolves rapidly by horizontal gene transfer (HGT). Here, we aim at understanding this apparent contradiction by studying the covariation between the repertoire of capsule genes and MGEs in approximately 4,000 genomes of Klebsiella pneumoniae (Kpn). We show that capsules drive phage-mediated gene flow between closely related serotypes. Such serotype-specific phage predation also explains the frequent inactivation of capsule genes, observed in more than 3% of the genomes. Inactivation is strongly epistatic, recapitulating the capsule biosynthetic pathway. We show that conjugative plasmids are acquired at higher rates in natural isolates lacking a functional capsular locus and confirmed experimentally this result in capsule mutants. This suggests that capsule inactivation by phage pressure facilitates its subsequent reacquisition by conjugation. Accordingly, capsule reacquisition leaves long recombination tracts around the capsular locus. The loss and regain process rewires gene flow toward other lineages whenever it leads to serotype swaps. Such changes happen preferentially between chemically related serotypes, hinting that the fitness of serotype-swapped strains depends on the host genetic background. These results enlighten the bases of trade-offs between the evolution of virulence and multidrug resistance and caution that some alternatives to antibiotics by selecting for capsule inactivation may facilitate the acquisition of antibiotic resistance genes (ARGs).

A study of how the complex interaction between capsules and mobile genetic elements shapes gene flow in populations of Klebsiella pneumoniae reveals that capsule inactivation by phage pressure facilitates its subsequent re-acquisition by conjugation, and this loss and re-gain process influences the gene flow towards other lineages whenever it leads to serotype changes.     

Interchangeability of the adsorption proteins of bacteriophages Ff and IKe.

The wild-type adsorption protein (g3p) of filamentous phage IKe cannot be exchanged with its analogous protein in the related Ff (M13, fd, and f1) phage particles. Deletion mutants of the protein, however, are assembled into Ff phage particles. These hybrid Ff phage particles bearing deleted IKe g3p attach to N pili, thus conserving the host attachment property of the protein but not its infection-initiating function. This means that the attachment specificity is determined by IKe g3p independently of other phage components in contact with it. Infection initiation function, the process in which phage DNA is released into the host, in contrast seems to require either more complex structural features of the protein (for example, a certain oligomeric structure) provided only in the original particle, or a concerted action of g3p with another particle component, not replaceable by its homologous counterpart in the related phage.     

Escherichia coli tolQ mutants are resistant to filamentous bacteriophages that adsorb to the tips, not the shafts, of conjugative pili

The tolQ (previously fii) mutation in Escherichia coli K12 inhibits infection by filamentous bacteriophages f1 and IKe but not by RNA-containing phage f2. This work extends these observations to other plasmid-specific bacteriophages including various filamentous. RNA-containing, and lipid-containing isolates. Only tip-adsorbing filamentous phages were affected by tolQ and not shaft-adsorbing ones. Electron microscopy showed that RP4-specific filamentous phage Pf3 was one of the latter kind. Several tip-adsorbing filamentous phages inhibited conjugation between tolQ strains carrying their specific plasmids, implicating the phage receptors (conjugative pili) as mating organelles. tolQ mutant strains were as proficient as their parents in conjugation mediated by a wide range of plasmids.     

Phages C-2 and J: IncC and IncJ plasmid-dependent phages, respectively

Phages C-2 and J were isolated from sewage. Phage C-2 was filamentous and formed plaques on Salmonella typhimurium strains carrying various C plasmids. It also plated on Proteus mirabilis and Serratia marcescens strains carrying particular C plasmids, but failed to form plaques on lines of Escherichia coli K12 strains harbouring most of these plasmids, although in all cases, phage multiplication on the strains was demonstrated. No phage increase occurred in any strain which lacked a C plasmid or contained plasmids of other incompatibility groups. The phage was sensitive to chloroform and, unlike other filamentous bacterial viruses, adsorbed to shafts of conjugative pili. It had a disc-like structure at the end which attached to the pilus. Phage C-2 had a buoyant density of 1 . 30 g cm-3 and a single-stranded circular DNA genome of 3 . 0 MDal. Phage J had an hexagonal head with an inter-apical distance of 40 nm and a short noncontractile tail. It was resistant to chloroform and diethyl ether. The phage formed plaques or propagated on E. coli strains harbouring some IncC plasmids and all IncJ and IncD plasmids tested. The phage did not form plaques but propagated on P. mirabilis and Ser. marcescens strains carrying these plasmids. It did not plate or propagate on S. typhimurium strains harbouring the plasmids. The plaques were very hazy and variable in size. The phage attached sparsely, at a site which appeared to be located at the base of the tail, to sides of conjugative pili.     

An activation domain of plasmid R1 TraI protein delineates stages of gene transfer initiation

Bacterial conjugation is a form of type IV secretion that transports protein and DNA to recipient cells. Specific bacteriophage exploit the conjugative pili and cell envelope spanning protein machinery of these systems to invade bacterial cells. Infection by phage R17 requires F-like pili and coupling protein TraD, which gates the cytoplasmic entrance of the secretion channel. Here we investigate the role of TraD in R17 nucleoprotein uptake and find parallels to secretion mechanisms. The relaxosome of IncFII plasmid R1 is required. A ternary complex of plasmid oriT, TraD and a novel activation domain within the N-terminal 992 residues of TraI contributes a key mechanism involving relaxase-associated properties of TraI, protein interaction and the TraD ATPase. Helicase-associated activities of TraI are dispensable. These findings distinguish for the first time specific protein domains and complexes that process extracellular signals into distinct activation stages in the type IV initiation pathway. The study also provided insights into the evolutionary interplay of phage and the plasmids they exploit. Related plasmid F adapted to R17 independently of TraI. It follows that selection for phage resistance drives not only variation in TraA pilins but diversifies TraD and its binding partners in a plasmid-specific manner.     

Functional identification of conjugation and replication regions of the tetracycline resistance plasmid pCW3 from Clostridium perfringens

Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.     

The unique conjugation system of IncHI3 plasmid MIP233

The conjugation system of the IncHI3 plasmid MIP233 was studied using a transfer-derepressed Tn5-insertion mutant. The conjugative pili of this plasmid were short pointed rods resembling rigid pili, with a well-defined modal length. Unlike plasmids with rigid pili, the MIP233 mutant mediated a surface + liquid conjugation system. The pili were serologically different from all known pilus types including H pili, and did not act as receptors for any known pilus-specific bacteriophage. They converted the surface conjugation system of RP4 to a surface + liquid one. Antiserum to pili of the mutant plasmid inhibited transfer of the wild-type plasmid MIP233, demonstrating that it contained only one transfer system.     

Examination of the phosphate in conjugative F-like pili by use of electron spectroscopic imaging.

F-like pili specified by conjugative plasmids have been reported to contain phosphate which may be noncovalently incorporated into the pilus. Electron spectroscopic imaging was able to detect phosphate in the filamentous, single-stranded DNA phage f1, used as positive control, but could not detect phosphate in F-like pili. Thus, the phosphate in purified pili which has been reported is probably derived from contaminating cell envelope material.     

Towards a structural biology of bacterial conjugation

Horizontal DNA transfer among bacteria (conjugation) requires the formation of secure intercellular contacts before DNA transfer can occur. The formation of such contacts among Gram-negative bacteria is mediated by conjugative pili. Like other pili, conjugative pili are filaments extending from the surface of donor cells. They are composed, in so far as is known, entirely of conjugative pilin subunits. Here, we review the structure of F-pilin, the subunit of which F-pili are composed. We emphasize recent studies suggesting a specific domain organization for F-pilin and present a model of how these domains might be arranged in filament subunits.     

细菌中整合性接合元件的研究进展

整合性接合元件是近年来在细菌中发现的一种可移动的基因元件,它位于染色体上,可通过接合转移的方式介导细菌间基因的水平转移。这种基因的水平转移有助于细菌适应特定的环境条件,但许多整合性接合元件包含耐药基因,这些遗传元件的水平转移极大地加速了耐药基因在同种及不同种属之间的传播,造成细菌的耐药以至多重耐药问题日益严重,耐药机制日趋复杂;同时整合性接合元件与基因岛有着密切的联系,因此对其特征及转移机制进行研究很有必要。     

Mutational analysis of F-pilin reveals domains for pilus assembly, phage infection and DNA transfer

The F-pilus has been implicated in recipient cell recognition during the establishment of a stable mating pair before conjugation as well as forming part of the conjugative pore for DNA transfer. The F-pilus is the site of attachment of the filamentous phages (M13, f1 and fd), which attach to the F-pilus tip, and the RNA phages, R17 and Qbeta, which attach to different sites exposed on the sides of the pilus. R17 has been shown to undergo eclipse, or capsid release, outside the cell on pili attached to cells. New and existing mutants of traA combined with natural variants of F-pilin were assayed for pilin stability and processing, pilus elongation, transfer, phage sensitivity and R17 eclipse. Phenotypes of these mutants indicated that the F-pilin subunit contains specific regions that can be associated with pilus assembly, phage sensitivity and DNA transport. Mutations involving lysines and phenylalanines within residues 45-60 suggest that these residues might participate in transmitting a signal down the length of the pilus that initiates DNA transfer or R17 eclipse.     

Transfer of genetic information from agrobacteria to bacterial and plant cells: membrane and supramembrane structures involved in transfer

The review deals with the supramembrane and membrane structures involved in the initial contact (attachment) of an agrobacterial cell with a bacterial or plant cell during the transfer of the agrobacterial genetic information. The relationships between the donor cell attachment to the recipient cell surface and the infection and conjugation processes are discussed. Experimental data on the recently found agrobacterial pili and surface protein rhicadhesin, which are involved in the conjugative transfer of the plasmid between agrobacteria, are considered. The role of adhesive and conjugative pili of E. coli in the initial and tight contacts is analyzed in the context of the recently proved similarity between the mechanisms of agrobacterial transformation in plants and conjugative transfer in bacteria. Possible involvement of the pilus in the conjugative transfer of agrobacterial DNA across the membranes of donor and recipient (bacterial and plant) cells is discussed.     

Mobility of Plasmids

Summary: Plasmids are key vectors of horizontal gene transfer and essential genetic engineering tools. They code for genes involved in many aspects of microbial biology, including detoxication, virulence, ecological interactions, and antibiotic resistance. While many studies have decorticated the mechanisms of mobility in model plasmids, the identification and characterization of plasmid mobility from genome data are unexplored. By reviewing the available data and literature, we established a computational protocol to identify and classify conjugation and mobilization genetic modules in 1,730 plasmids. This allowed the accurate classification of proteobacterial conjugative or mobilizable systems in a combination of four mating pair formation and six relaxase families. The available evidence suggests that half of the plasmids are nonmobilizable and that half of the remaining plasmids are conjugative. Some conjugative systems are much more abundant than others and preferably associated with some clades or plasmid sizes. Most very large plasmids are nonmobilizable, with evidence of ongoing domestication into secondary chromosomes. The evolution of conjugation elements shows ancient divergence between mobility systems, with relaxases and type IV coupling proteins (T4CPs) often following separate paths from type IV secretion systems. Phylogenetic patterns of mobility proteins are consistent with the phylogeny of the host prokaryotes, suggesting that plasmid mobility is in general circumscribed within large clades. Our survey suggests the existence of unsuspected new relaxases in archaea and new conjugation systems in cyanobacteria and actinobacteria. Few genes, e.g., T4CPs, relaxases, and VirB4, are at the core of plasmid conjugation, and together with accessory genes, they have evolved into specific systems adapted to specific physiological and ecological contexts.     

Phage tf-1: a filamentous bacteriophage specific for bacteria harbouring the IncT plasmid pIN25

Phage tf-1 is a filamentous phage which is about 800 nm in length, 10 nm in width and has slightly tapered ends. The phage was isolated from sewage and formed plaques or propagated only on Escherichia coli, Salmonella typhimurium and Klebsiella oxytoca strains harbouring the IncT plasmid pIN25 at 30 degrees C. It adsorbed in large numbers to pIN25-encoded long thick flexible conjugative pili formed at 30 degrees C and also to the short form of these pili synthesized at 37 degrees C. The reason for the failure to form plaques at 37 degrees C is not known. The adsorption site is a short length of the pilus shaft extending 100-200 nm back from the distal tip. Efficient phage tf-1 adsorption to the same site was found for pili determined by other IncT plasmids in spite of the fact that phage tf-1 did not plate or propagate on strains harbouring them. However, areas of specific partial clearing on lawns of these plasmid-containing bacteria were produced by phage in high concentrations. Lack of plaque-formation could be due to inefficient intracellular assembly coupled to avid adsorption of any liberated phage to pili. The phage differs from all but one other filamentous phage by being sensitive to diethyl ether.     

Role of F Pili in the Penetration of Bacteriophage fl

Early stages of infection of Escherichia coli with the filamentous bacteriophage f1 were examined in the electron microscope. Purified phage-bacteria complexes were prepared at various time intervals after the initiation of synchronous infection. Cells were scored for the total number of F pili, the number of F pili with f1 attached, the number of intact phage particles which occurred at the surface of the cell, and F pilus length. Electron microscope autoradiographs were also prepared at each time interval. The results showed that the average number of F pili with f1 attached decreased with time as phage deoxyribonucleic acid (DNA) entered the cell. Concomitant with this loss, the remaining F pili became shorter. The rate of entry of phage DNA into the cell followed, with a short lag, the rate of loss of F pili with f1 attached. During the lag period, intact phage particles accumulated at the surface of the cell. The results from radioautographs showed that no phage DNA could be located within the F pilus. These results suggest that F pili are resorbed by the cell during infection with the bacteriophage f1. Parallel experiments with noninfected cultures further suggest that pilus resorption may be a normal cellular phenomenon.     

Bacteriophage X-2: a filamentous phage lysing IncX-plasmid-harbouring bacterial strains

Phage X-2, a filamentous rod about 950 nm in length, was isolated from sewage as plating on strains of Escherichia coli, Salmonella typhimurium or Serratia marcescens carrying either the IncX plasmid R6K, or the unique plasmid R775. Phage X-2 differs morphologically from a previously described very broad host range filamentous phage X which also lyses plasmid R6K-carrying strains and the phages differ in their resistance to inactivation by diethyl ether. Phage X-2 is serologically unrelated to phage X and the X-like phages IKe and I2-2. The adsorption site of the phage on the plasmid-bearing strains could not be determined but evidence implicating conjugative pili is presented.     

Conjugative transfer can be inhibited by blocking relaxase activity within recipient cells with intrabodies

Horizontal transfer of antibiotic resistance genes carried by conjugative plasmids poses a serious health problem. As conjugative relaxases are transported to recipient cells during bacterial conjugation, we investigated whether blocking relaxase activity in the recipient cell might inhibit conjugation. For that purpose, we used an intrabody approach generating a single-chain Fv antibody library against the relaxase TrwC of conjugative plasmid R388. Recombinant single-chain Fv antibodies were engineered for cytoplasmic expression in Escherichia coli cells and either selected in vitro for their specific binding to TrwC, or in vivo by their ability to interfere with conjugation using a high-throughput mating assay. Several intrabody clones were identified showing specific inhibition against R388 conjugation upon cytoplasmic expression in the recipient cell. The epitope recognized by one of these intrabodies was mapped to a region of TrwC containing Tyr-26 and involved in the conjugative DNA-processing termination reaction. These findings demonstrate that the transferred relaxase plays an important role in the recipient cell and open a new approach to identify specific inhibitors of bacterial conjugation.     

Tanzawaic Acids,a Chemically Novel Set of Bacterial Conjugation Inhibitors

Bacterial conjugation is the main mechanism for the dissemination of multiple antibiotic resistance in human pathogens. This dissemination could be controlled by molecules that interfere with the conjugation process. A search for conjugation inhibitors among a collection of 1,632 natural compounds, identified tanzawaic acids A and B as best hits. They specially inhibited IncW and IncFII conjugative systems, including plasmids mobilized by them. Plasmids belonging to IncFI, IncI, IncL/M, IncX and IncH incompatibility groups were targeted to a lesser extent, whereas IncN and IncP plasmids were unaffected. Tanzawaic acids showed reduced toxicity in bacterial, fungal or human cells, when compared to synthetic conjugation inhibitors, opening the possibility of their deployment in complex environments, including natural settings relevant for antibiotic resistance dissemination.