Cellular location and temperature-dependent assembly of IncHI1 plasmid R27-encoded TrhC-associated conjugative transfer protein complexes.

Conjugal transfer of IncHI plasmid DNA between Gram-negative bacteria is temperature sensitive, as mating is optimal between 22 degrees C and 30 degrees C but is inhibited at 37 degrees C. R27, isolated from Salmonella enterica serovar Typhi, is an IncHI1 plasmid of 180 kbp that has been sequenced completely. The gene encoding green fluorescent protein (GFP) was inserted into R27 in frame with trhC. TrhC is a mating pair formation (Mpf) protein that is essential for plasmid transfer and H-pilus production. Fluorescence microscopy allowed visualization of the TrhC-GFP fusion protein, and Escherichia coli cells were examined for the subcellular localization and temperature-dependent production of TrhC-GFP. At 27 degrees C, TrhC-GFP was found at the periphery of cells as discrete foci, indicating an association of TrhC within protein complexes in the bacterial cell membrane, whereas at 37 degrees C, little fluorescence was detected. These foci probably represent the intracellular position of protein complexes involved in conjugative transfer, as the formation of foci was dependent upon the presence of other Mpf proteins. During temperature shift experiments from 37 degrees C to 27 degrees C, a long lag period was required for generation of GFP foci. Conversely, during short shifts from 27 degrees C to 37 degrees C, the GFP foci remained stable. These results suggest that the expression of transfer genes in the Tra2 region of R27 is temperature dependent. Subcellular localization of TrhC was verified by cellular fractionation. Expression patterns of TrhC-GFP were confirmed with immunoblot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). These results allow us to propose mechanisms to explain the temperature-sensitive transfer of R27.

Sequencing and characterization of Salmonella typhi plasmid R27 (incompatibility group HI1) trhC, a transfer gene encoding a potential nucleoside triphosphate-binding domain.

IncHI1 plasmids are one of the few plasmids known to mediate multiple antibiotic resistance in Salmonella typhi. These plasmids are temperature-sensitive for transfer and R27 is the prototype plasmid. DNA sequencing within the Tra2 region of R27, encoding genes involved in mating pair formation, identified trhC encoding the TrhC protein of 90,000 Da, which was visualized using an in vitro transcription/translation system. Expression of the TrhC protein also identified two smaller protein products of approximately 23 and 25 kDa which are predicted to be protease digestion products. The migration of these smaller products changed when the reactions were run at 28 vs 37 degrees C. The TrhC protein contained a putative nucleotide triphosphate-binding protein and exhibited sequence similarities with several other proteins implicated in bacterial conjugation, including TraC (F), TraB (pKM101), VirB4 (Ti), TrbE (RP4). Phylogenetic analysis showed TrhC was most closely related to TraC. Mini-Tn10 insertions in trhC generated transfer defective mutants, and no pili were specified by the trhC mutants. The trhC gene appeared to be a hot spot for transposon insertion as 37.5% mapped into this open reading frame. One trhC mutant, pDT2990, was able to be complemented by a cloned trhC gene giving a transfer frequency of 1 x 10(-3) transconjugants per recipient in an 18-h mating, whereas the wild-type transfer frequency of R27 was 1 x 10(-2) transconjugants per recipient.

Molecular analysis of RepHI1A, a minimal replicon of the IncHI1 plasmid R27.

R27, a large conjugative plasmid of the HI incompatibility group, was subjected to a subcloning analysis which revealed the presence of a Poll-independent replicon and determinants contributing to incompatibility within a 2.7 kb SalI/XbaI fragment. The DNA sequence of the minimal replicon revealed the presence of a large open reading frame (ORF) as well two sets of 19 bp repeated oligonucleotides (iterons), in addition to characteristic Escherichia coli origin elements. The protein encoded by the ORF possesses homology with replication initiator proteins encoded by a number of plasmids from different incompatibility groups. Deletion analysis suggested that the iterons are responsible for incompatibility reactions. Dissection of the replicon confirmed this and defined a minimal origin of 230 bp. The putative replication initiator was expressed in an in vitro transcription-translation system, and the 5' end of the mRNA encoding its synthesis was identified. Transcriptional fusion of the repA promoter to lacZ demonstrated an autoregulatory function of RepA. A series of iterons present downstream of the RepA coding sequence are dispensable but are responsible for copy-number control. The minimal replicon appears to be partition-defective.

Isolation and location on the R27 map of two replicons and an incompatibility determinant specific for IncHI1 plasmids.

Two replicons were isolated independently from different IncHI1 plasmids. One was isolated from R27, and a second was isolated from pIP522. We demonstrate, by DNA-DNA hybridization experiments, that these maintenance regions are different and that they are specific to, and carried by, all IncHI1 plasmids tested. In view of this specificity we decided to designate the replicon isolated from R27 as RepHI1A and the replicon isolated from pIP522 as RepHI1B. These two autoreplicative regions are not related to a third replicon present in all IncHI1 plasmids that bears homology with RepFIA and that expresses the characteristic incompatibility of IncHI1 subgroup plasmids toward F factor (D. Saul, D. Lane, and P. L. Bergquist, Mol. Microbiol. 2:219-225, 1988; D. E. Taylor, R. W. Hedges, and P. L. Bergquist, J. Gen. Microbiol. 131:1523-1530, 1985). These results demonstrate that all IncHI1 plasmids tested contain at least three replicons. An incompatibility (Inc) region that hybridizes specifically to all the IncHI1 plasmids was previously isolated (M. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Although this Inc locus is not located in an autoreplicative region of IncHI1 plasmids, we observed that this locus stabilizes a low-copy-number replicon. This Inc locus is probably a component of an active partition locus involved in the maintenance of IncHI1 plasmids. The nucleotide sequence of the Inc region contains direct repeats of 31 bp. In addition, this incompatibility determinant hybridizes specifically with IncHI1 plasmids but expresses incompatibility toward plasmids of both IncHI subgroups (IncHI1 and IncHI2). In this communication, we present the mapping of these maintenance elements on the R27 genome.

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.

The choice between analysis of variance and analysis of covariance with special reference to the analysis of organ weights in toxicology studies.

The problem of when to include a covariate in the analysis of variance is considered in the special case of organ weight analysis in animal toxicology studies. The covariate is bodyweight prior to death, which may be subject to treatment effects. A simulation study is carried out to compare four rules for deciding whether or not to include the covariate. It is concluded that if there is background information which shows a linear relationship between variate and covariate it is advisable to adjust for the covariate, however weak the relationship may appear to be on the current set of data. Alternative procedures lead to unacceptably high Type I error rates.