A subassembly of R27-encoded transfer proteins is dependent on TrhC nucleoside triphosphate-binding motifs for function but not formation

The transfer of plasmid DNA molecules between bacterial cells is achieved by a large array of conjugative transfer proteins which assemble into both cytoplasmic and membrane-associated complexes. TrhC is a membrane-associated protein that is required for the transfer of the IncHI1 resistance plasmid R27. Homologous proteins are encoded in all known conjugative systems, and each contains characteristic nucleoside triphosphate (NTP)-binding domains. An assembly of R27-encoded proteins was previously visualized by use of a TrhC-green fluorescent protein fusion, which appeared as discrete membrane-associated fluorescent foci. We have utilized this experimental system to determine the requirements for assembly of this TrhC-associated protein complex, and we found that 12 of the other 18 R27 transfer proteins are required for focus formation. An individual focus possibly represents a subassembly comprised of some or all of these transfer proteins. These data support the notion that the transfer apparatus is a multicomponent structure. In contrast, substitutions and deletions within TrhC NTP-binding motifs had minor effects on focus formation, but these mutations did affect plasmid transfer and bacteriophage susceptibility. These results indicate that TrhC requires intact NTP-binding motifs to function during conjugative transfer but that these motifs are not essential for the assembly of TrhC into a complex with other transfer proteins.     

Interaction between the IncHI1 plasmid R27 coupling protein and type IV secretion system: TraG associates with the coiled-coil mating pair formation protein TrhB

Assemblies of plasmid-encoded proteins direct the conjugative transfer of plasmid DNA molecules between bacteria. These include the membrane-associated mating pair formation (Mpf) complex necessary for pilus production and the cytoplasmic relaxosome required for DNA processing. The proposed link between these distinct protein complexes is the coupling protein (the TraG family of proteins). Interactions between the coupling protein and relaxosome components have been previously characterized and we document here, for the first time, a direct interaction between the coupling protein and an Mpf protein. Using the adenylate cyclase bacterial two-hybrid (BTH) system, we present in vivo evidence that the IncHI1 plasmid R27-encoded proteins TraG and TrhB interact. This interaction was verified through a co-immunoprecipitation reaction. We have also been able to delineate the interaction domain of TrhB to TraG by showing a positive interaction using the first 220 amino acids of TrhB (452 aa). TrhB has a proline-rich domain from amino acids 135-173 which may serve to facilitate protein interactions and/or periplasmic extension. TrhB self association was detected using far-Western, co-immunoprecipitation, and also BTH analysis, which was used to define the homotypic interaction domain, comprising a predicted coiled-coil region at residues 77-124 of TrhB. These data support a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane Mpf complex.     

Functional and mutational analysis of conjugative transfer region 1 (Tra1) from the IncHI1 plasmid R27

The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.     

H-pilus assembly kinetics determined by electron microscopy.

The kinetics of pilus outgrowth were examined for Escherichia coli containing pDT1942, a TnlacZ insertion derivative of the IncHI1 plasmid R27, which was derepressed for transfer. IncHI1 plasmids are thermosensitive for transfer. The pili specified by pDT1942 were examined by transmission electron microscopy after the pilus had been labeled with the H-pilus-specific bacteriophage Hgal, which had been inactivated with RNase A. H pili were extended by extrusion from the cell surface and not by the addition of pilin subunits to the pilus tip. After pili were removed by vortexing, the outgrowth of full-length pili (2 microns long) required 20 min. H pili expressed at the transfer optimal temperature (27 degrees C) remained stable after incubation at the transfer inhibitory temperature (37 degrees C), but the formation of mating aggregates was inhibited at 37 degrees C. Within 1 min of exposure of the host cell to a heat stimulus of 50 degrees C, pili vanished. Pili were observed in straight and flexible forms with a field emission scanning electron microscope, which may indicate a dynamic role for the pilus in conjugation.     

pIN32: a cointegrate plasmid with IncHI2 and IncFII components

An Enterobacter cloacae strain isolated from the faeces of a child with diarrhoea in Indonesia contained a transferable 216 MDa plasmid, pIN32, exhibiting IncHI2 phenotypic characters, including temperature sensitivity of transfer and the expression of H serotype pili at a repressed level. A derivative plasmid (pIN32-1), which had lost the IncHI2 phenotype, and contained only 60 MDa of the original replicon, was obtained after mating at 37 degrees C. It was IncFII, showed regions of homology with plasmid R100, determined IncFII serotype conjugative pili constitutively and was transfer-derepressed. After overnight growth at 37 degrees C in non-selective medium, pIN32 gave rise to another derivative, pIN32-2 (size 184.3 MDa), which retained the IncHI2 phenotype and several other pIN32 characters.     

Genetic analysis of transfer and incompatibility functions within the IncHI plasmid R27

This study was undertaken to establish a transfer complementation system for IncH plasmids and to locate regions of incompatibility within the HI1 plasmid, R27. Two regions of R27 were found to contribute to incompatibility as determined by incompatibility testing with fragments of R27 cloned in cosmid vectors. One of these regions hybridized with the IncHI1 rep probe (Couturier et al., Microbiol. Rev. 52, 375-395, 1988). Complementation analysis was carried out using transfer-deficient mutants of R27 in combination with pHH1508a. Cosmid vectors, which contained cloned restriction fragments of R27, were able to complement selected R27 Tra- mutants, enabling the transfer-deficient plasmid to transfer at near-normal frequencies. Complementation of R27 Tra- plasmids by pHH1508a at both 26 and 37 degrees C was shown to occur, but was host-dependent in its degree. These results suggest that the transfer mechanisms of IncHI and IncHII plasmids are related.     

TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.     

Effect of lipopolysaccharide mutations on recipient ability of Salmonella typhimurium for incompatibility group H plasmids.

Previous investigations of the incompatibility group F, P, and I plasmid systems revealed the important role of the outer membrane components in the conjugal transfer of these plasmids. We have observed variability in transfer frequency of three incompatibility group H plasmids (IncHI1 plasmid R27, IncHI2 plasmid R478, and a Tn7 derivative of R27, pDT2454) upon transfer into various Salmonella typhimurium lipopolysaccharide (LPS) mutants derived from a common parental strain, SL1027. Recipients with truncated outer core via the rfaF LPS mutation increased the transfer frequency of the IncH plasmids by up to a factor of 10(3). Mutations which resulted in the truncation of the residues following 3-deoxy-D-manno-octulosonic acid, such as the rfaE and rfaD mutations, decreased the transfer frequency to undetectable levels. Addition of phosphorylethanolamine, a component of wild-type LPS, to the media decreased the frequency of transfer of R27 into wild-type and rfaF LPS mutant recipients tested. Reversing the direction of transfer, by mating LPS mutant donors with wild-type recipients, did not affect the frequency of transfer compared to the standard matings of wild-type donor with LPS mutant recipient. These findings demonstrate that conjugation interactions affected by LPS mutation are not specific for the recipient cell. Our results suggest that LPS mutation does not affect conjugation via altered pilus binding but affects some later steps in the conjugative process, and alteration of transfer frequency by O-phosphorylethanolamine and LPS truncation is due to charge-related interactions between the donor and recipient cell.     

Characterization of the Tra2 Region of the IncHI1 Plasmid R27

In this study, the DNA sequence of one of the transfer regions of the IncHI1 plasmid R27 was determined. This region, which corresponds to coordinates 0-40 on the R27 map has been called the Tra2 region, and is believed to be involved in mating pair formation. DNA sequence analysis of the transfer region identified 11 open reading frames which showed similarities to the transfer genes from other conjugative systems. The R27 transfer genes appear to most closely resemble the genes from the F plasmid and Sphingomonas aromaticivorans plasmid pNL1, both within the individual genes and in the overall gene order. The Tra2 region is also distinct in that replication, partitioning, and stability genes are found in the middle of the transfer region. The R27 Tra2 region also contains a gene, trhF, which appears to be related to the TraF genes of Agrobacterium and Rhizobium species. This, along with the temperature-sensitive transfer system found in both H plasmids and Agrobacterium, leads to the speculation that the R27 transfer region evolved from both ancestral F-like and P-like plasmids.     

Transfer-defective and tetracycline-sensitive mutants of the incompatibility group HI plasmid R27 generated by insertion of transposon 7

Transposon Tn7 insertion was used to obtain either transfer-defective (Tra-) or tetracycline-sensitive (Tc-) mutants of the HI incompatibility group (IncHI) plasmid R27. The 600 apparent R27::Tn7 derivatives fell into three classes: Tra-, Tc-, and Tra- Tc-. Mutants of R27 defective in the thermosensitive mode of transfer characteristic of IncH plasmids were obtained with transfer frequencies of less than 1 X 10(-8) transconjugants per recipient after 18 hr at 26 degrees C. These mutants, which were generated at a frequency of 1 per 100 insertions, were nonleaky and nonrevertible. Tc- mutants of R27, generated at a frequency of 0.5 per 100 insertions, were also nonrevertible. Loss of tetracycline resistance was associated with an increased frequency of transfer (average 3.6 X 10(-3) transconjugants per donor per hour at 30 degrees C) compared with transfer of the wild-type R27 plasmid (1.6 X 10(-8) per donor per hour). Tn7 insertions which generated Tc- or Tra- mutants of R27 had no effect on entry exclusion of other H group plasmids. The molecular weights of Tra- and Tc- R27::Tn7 derivatives were approximately 120.5 MDa, corresponding to the sum of R27 (112 MDa) and Tn7 (8.5 MDa). A third class of Tn7 insertion derivatives (Tra- Tc-) was obtained; however, strains expressing this phenotype were plasmid free, and appeared to have Tn7 integrated at a chromosomal site. Restriction digestion with XbaI and subsequent hybridization with ColE1::Tn7 were used to compare R27::Tn7 derivatives and to locate Tn7 insertion sites. Loss of tetracycline resistance was associated with Tn7 insertion into a 24-kb XbaI fragment of R27. Although loss of plasmid transfer in several R27::Tn7 derivatives was accompanied by insertion of Tn7 into a 14-kb XbaI fragment of the plasmid, these mutants had also undergone a small increase in the size of the 24-kb XbaI fragment of R27.     

Characterization of the double-partitioning modules of R27: correlating plasmid stability with plasmid localization

Plasmid R27 contains two independent partitioning modules, designated Par1 and Par2, within transfer region 2. Par1 is member of the type I partitioning family (Walker-type ATPase), and Par2 is a member of the type II partitioning family (actin-type ATPase). Stability tests of cloned Par1 and Par2 and insertional disruptions of Par1 and Par2 within R27 demonstrated that Par1 is the major stability determinant whereas Par2 is the minor stability determinant. Creation of double-partitioning mutants resulted in R27 integrating into the chromosome, suggesting that at least one partitioning module is required for R27 to exist in the extrachromosomal form. Using the lacO/LacI-green fluorescent protein (GFP) system, we labeled and visualized R27 and R27 partitioning mutants (Par1(-) and Par2(-)) under different growth conditions in live Escherichia coli cells. Plasmid R27 was visualized as the discrete GFP foci present at the mid- and quarter-cell regions in >99% of the cells. Time lapse experiments demonstrated that an increase in R27 plasmid foci resulted from focus duplication in either the mid- or quarter-cell regions of E. coli. Both R27 Par(-) variants gave a high percentage of plasmidless cells, as suggested by a uniform GFP signal, and cells with GFP patterns scattered throughout the entire cell, suggesting that plasmid molecules are randomly distributed throughout the cytoplasm. Those cells that did contain R27 Par(-) with one or two discrete foci had localization patterns that were statistically different from those formed with wild-type R27. Therefore, these results suggest that partitioning-impaired plasmids are characterized by individual and clustered plasmids that are randomly located within the host cytoplasm.     

Functional and mutational analysis of conjugative transfer region 2 (Tra2) from the IncHI1 plasmid R27

The transfer 2 region (Tra2) of the conjugative plasmid drR27 (derepressed R27) was analyzed by PSI-BLAST, insertional mutagenesis, genetic complementation, and an H-pilus assay. Tra2 contains 11 mating-pair formation (Mpf) genes that are essential for conjugative transfer, 9 of which are essential for H-pilus production (trhA, -L, -E, -K, -B, -V, -C, -P, and -W). TrhK has similarity to secretin proteins, suggesting a mechanism by which DNA could traverse the outer membrane of donors. The remaining two Mpf genes, trhU and trhN, play an auxiliary role in H-pilus synthesis and are proposed to be involved in DNA transfer and mating-pair stabilization, respectively. Conjugative transfer abilities were restored for each mutant when complemented with the corresponding transfer gene. In addition to the essential Mpf genes, three genes, trhO, trhZ, and htdA, modulate R27 transfer frequency. Disruption of trhO and trhZ severely reduced the transfer frequencies of drR27, whereas disruption of htdA greatly increased the transfer frequency of wild-type R27 to drR27 levels. A comparison of the essential transfer genes encoded by the Tra2 and Tra1 (T. D. Lawley, M. W. Gilmour, J. E. Gunton, L. J. Standeven, and D. E. Taylor, J. Bacteriol. 184:2173-2183, 2002) of R27 to other transfer systems illustrates that the R27 conjugative transfer system is a chimera composed of IncF-like and IncP-like transfer systems. Furthermore, the Mpf/type IV secretion systems encoded by IncH and IncF transfer systems are distinct from that of the IncP transfer system. The phenotypic and ecological significance of these observations is discussed.     

Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10

Chloramphenicol resistance in Salmonella typhi is medicated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, Apal, Xbal, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer.     

Nucleolar localization of Aspergillus fumigatus CgrA is temperature-dependent

Pathogenic fungi must adapt to multiple adverse environmental conditions during the transition from the environment to a mammalian host, one of which is temperature. The ability of Aspergillus fumigatus to grow optimally under conditions of thermal stress requires the nucleolar protein CgrA. In this study, we have determined how temperature affects the intracellular localization of CgrA in A. fumigatus using a green fluorescent protein (GFP)-tagging approach. At 22 degrees C, CgrA was almost exclusively in the nucleolus, with a ratio of nucleolar to cytoplasmic fluorescence of 10:1. At 37 degrees C, the ratio of nucleolar to cytoplasmic fluorescence was reduced fivefold, and increased correspondingly in the cytoplasm. This effect was not seen with the nucleolar protein NopA in wild-type A. fumigatus. However, in a DeltacgrA mutant NopA was delocalized from the nucleolus at 37 degrees C but not at 22 degrees C. These results provide evidence for a temperature-dependent mechanism of intracellular localization for CgrA, and suggest that CgrA facilitates nucleolar compartmentalization of NopA at higher temperature.     

Genetic and nucleotide sequence analysis of the gene htdA, which regulates conjugal transfer of IncHI plasmids.

IncHI plasmids are naturally repressed for conjugative transfer and do not allow efficient propagation of the IncH pilus-specific phage Hgal. Transposons Tn7, Tn5, and TnlacZ were inserted into IncHI plasmids R478, R477-1, and R27, respectively, leading to the isolation of several plasmid mutants which exhibited increased levels of transfer and also permitted good lysis with phage Hgal. A 4.3-kb HindIII fragment from R478 reversed both phenotypic effects of derepression for the R477-1::Tn5 and the R478::Tn7 derivatives, pKFW99 and pKFW100, respectively. Exonuclease III deletions of this fragment and nucleotide sequence analysis indicated that the gene responsible for transfer repression, named here htdA, encoded a polypeptide of 150 amino acids. Cloning and sequence analysis of pDT2454 (R27::TnlacZ) revealed that the transposon had inserted into an open reading frame (ORF) which had an 83% amino acid identity with the R478 htdA gene. Maxicell analysis showed both the R27 and R478 HtdA products had molecular masses of 19.9 kDa. Conjugation experiments showed that the cloned htdA determinants caused a significant reduction of the transfer frequencies of wild-type R478 and R27 plasmids. Examination of both R478 derepressed mutants, pKFW100 and pKFW101, indicated that both transposon insertions occurred upstream of the htdA ORF. The results suggest that HtdA is a regulatory component of IncH plasmid transfer and also show that the region upstream of the htdA ORF is involved in transfer repression. The locations of the htdA determinants were identified on the plasmid maps of R27 and R478.     

Rifampin disrupts conjugal and chromosomal deoxyribonucleic acid metabolism in Escherichia coli K-12 carrying some IncIalpha plasmids.

The effects of rifampin and chloramphenicol on the transfer of ColIdrd-1 have been examined to determined whether transfer requires the synthesis of an untranslated species of ribonucleic acid (RNA), as proposed in models for the transfer of another IncIalpha plasmid, R64drd-11. When RNA synthesis was inhibited throughout mating by rifampin, ColI transfer between dna+ bacteria occurred at the normal rate for about 10 min and then stopped abruptly. Conjugational deoxyribonucleic acid (DNA) synthesis in dnaB mutants indicates that plasmid DNA was made in the rifampin-treated donors to replace the transferred material but the DNA tended to be unstable. In the presence of chloramphenicol, transfer of ColI gradually diminished over a longer period. Rifampin, but not chloramphenicol, was found to have unpredicted effects on chromosomal DNA metabolism in unmated dna+ and dnaB bacteria when they harbor any of three IncIalpha plasmids (ColIdrd-1, R144drd-3, and R64drd-11). Replication of the bacterial chromosome in such cells stopped abruptly about 15 min after the addition of rifampin, and at 41 degrees C, but not 37 degrees C, this was followed by extensive DNA breakdown. These findings suggest that curtailment of IncIalpha plasmid transfer by the drug results from a general disruption of DNA metabolism rather than from inhibition of a species of RNA essential for transfer.