Nucleotide sequence of the tra YALE region from IncFV plasmid pED208

Finlay, B. Brett; Frost, Laura S.; Paranchych, W.

doi:10.1128/jb.168.2.990-998.1986

Cited by 25 publications

(16 citation statements)

References 40 publications

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“…Southern hybridization showed the ardArelated sequence to be on the -23 kb Hind111 fragment which contains oriT and the pED208 leading region and is present in recombinant pBF105 (Finlay et al, 1986). The ardA hybridizing sequence was delimited further to a 11-5 kb EcoRI fragment, carried by pLG2053, and then to a 3.3 kb SphI-SaA fragment, carried by pLG2054 (Fig.…”

Section: Location and Nucleotide Sequence Of Arda On An Incw Plasmidmentioning

confidence: 99%

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Distribution of the ardA family of antirestriction genes on conjugative plasmids

Chilley

Wilkins

1995

Microbiology

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The ardA gene of I1 plasmid Collb-P9 was previously shown to alleviate DNA restriction by type I enzymes and to promote conjugative transmission of the unmodified plasmid to a restricting host. To clarify the ecological role of s d A , its distribution was determined on plasmids from 23 incompatibility groups using hybridization to the coding sequence as an assay. Hybridizing sequences, shown by nucleotide sequencing to have at least 60% identity with a d A , were detected on plasmids belonging to the I complex (IncB, I1 and K), the F complex (IncFV) and the lncN group. The a d A homologues were found to specify an antirestriction phenotype which was enhanced by genetic derepression of the plasmid transfer system. a d A loci map in plasmid leading regions but show no consistent association with a particular type of origin-oftransfer or a leading region gene of the ssb (single-stranded DNA-binding protein), psis (plasmid SOS inhibition) and hok (host killing) families. It may be significant that a d A + plasmids are authentic enterobacterial plasmids and that type I restriction systems are associated historically with members of the Enterobacteriaceae.

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Section: Location and Nucleotide Sequence Of Arda On An Incw Plasmidmentioning

confidence: 99%

“…IncN plasmid pKMlOl , which contains an additional second antirestriction gene called ardB (Belogurov e t al., 1992(Belogurov e t al., , 1993 (Finlay et al, 1986) were provided by L. Frost. pLG221 is ColIbdrd-1 cib: : Tn5 (Howland et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

Distribution of the ardA family of antirestriction genes on conjugative plasmids

Chilley

Wilkins

1995

Microbiology

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“…We assayed the specificity of the TraA–TraQ interaction by comparing the abilities of TraQ1 and TraQ5 to interact with F‐pilin and a related conjugative pilin in the yeast two‐hybrid assay (see Experimental procedures ). For the comparison, we chose pED208 pilin, which is 44% identical and 58% similar to F‐pilin (Finlay et al ., 1986). The two pilins are similarly organized into four structural domains, two of which, domains II and IV, are hydrophobic (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The evidence that TraQ interacts with domain IV of F‐pilin is also consistent with the observation that membrane insertion of TraA–PhoA fusion polypeptides in E. coli was facilitated by the presence of other tra genes in the same cells only when the TraA segment included domain IV (Paiva and Silverman, 1996). Finally, the observation that pED208 traA failed to interact with the F TraQ polypeptide in the yeast two‐hybrid assay could explain the failure of pED208 traA to complement an F traA mutant in E. coli (Finlay et al ., 1986).…”

Section: Discussionmentioning

confidence: 99%

Interaction between the F plasmid TraA (F‐pilin) and TraQ proteins

et al. 1999

Molecular Microbiology

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Elaboration of conjugative (F) pili by F+ strains of Escherichia coli requires the activities of over a dozen F-encoded DNA transfer (Tra) proteins. The organization and functions of these proteins are largely unknown. Using the yeast two-hybrid assay, we have begun to analyse binary interactions among the Tra proteins required for F-pilus formation. We focus here on interactions involving F-pilin, the only known F-pilus subunit. Using a library of F tra DNA fragments that contained all the F genes required for F pilus formation in a yeast GAL4 activation domain vector (pACTII), we transformed yeast containing a plasmid (pAS1CYH2traA) encoding a GAL4 DNA-binding domain-F-pilin fusion. Doubly transformed cells were screened for GAL4-dependent gene expression. This screen repeatedly identified only a single Tra protein, TraQ, previously identified as a likely F-pilin chaperone. The F-pilin-TraQ interaction appeared to be specific, as no transcriptional activation was detected in yeast transformants containing pACTIItraQ plasmids and the Salmonella typhi pED208 traA gene cloned in pAS1CYH2. Two traQ segments isolated in the screen against F-pilin were tested for complementation of a traQ null allele in E. coli. One, lacking the first 11 (of 94) TraQ amino acids, restored DNA donor activity, donor-specific bacteriophage sensitivity and membrane F-pilin accumulation to wild-type levels. The second, lacking the first 21 amino acids, was much less effective in these assays. Both TraQ polypeptides accumulated in E. coli as transmembrane proteins. The longer, biologically active segment was fused to the GAL4 DNA-binding domain gene of pAS1CYH2 and used to screen the tra fragment library. The only positives from this screen identified traA segments. The fusion sites between the traA and GAL4 segments identified the hydrophobic, C-terminal domain IV of F-pilin as sufficient for the interaction. As TraQ is the only Tra protein required for the accumulation of inner membrane F-pilin, the interaction probably reflects a specific, chaperone-like function for TraQ in E. coli. Attempts to isolate an F-pilin-TraQ complex from E. coli were unsuccessful, suggesting that the interaction between the two is normally transient, as expected from previous studies of the kinetics of TraA membrane insertion and processing to F-pilin.

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“…The sequence of ColB2 pilin is almost identical to that of F pilin, and the structure of the ColB2 pilus should also be identical (12). While the sequence of pED208 pilin is less homologous to that of F pilin (13), fiber diffraction has shown that the arrangement of subunits in the pilus is the same (19). If each subunit contained 1 to 2 mol of phosphate, the concentration of phosphate along the pilus would be 3.9 to 7.8 residues per nm, which is comparable to that found in the fl phage.…”

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

Frost

Bazett-Jones

1991

J Bacteriol

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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 fl, used as a 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.Self-transmissible plasmids in gram-negative bacteria specify filamentous, extracellular appendages called pili (16,30). These pili are essential for plasmid transfer and have been implicated in establishing cell-to-cell contact during formation of a mating bridge between the donor (plasmidcontaining) and recipient cells and in recognizing fellow donor cells, preventing redundant plasmid transfer, in a process known as surface exclusion. These pili are known to be the site of attachment for a wide range of bacteriophages; the majority of these phages are either isometric RNA phages or filamentous DNA phages. There are two attractive but speculative possibilities for a role for phosphate in pilus structure and function. One model suggests that a pilinbound phosphate is involved in signalling that a recipient cell or phage particle has attached to the pilus tip, while the second model suggests that it is involved in pilus assembly and retraction.Pilus outgrowth has been tied to the membrane potential (32) and is affected by a variety of procedures, including the addition of metabolic poisons and changes in temperature (25)(26)(27). Phage infection is similarly affected by altering the energy level in the cell (9, 33). Since the eclipse of RNA phage particles can occur at sites on an F pilus distal from the cell surface and is dependent on an energy source in the cell, the pilus may be able to deliver energy to points along its length (29). Purified pili do not have this property (17,31). If the pilin subunit is phosphorylated, it would be a good candidate for a role in the bioenergetics of pilus function.

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Nucleotide sequence of the tra YALE region from IncFV plasmid pED208

Cited by 25 publications

References 40 publications

Distribution of the ardA family of antirestriction genes on conjugative plasmids

Distribution of the ardA family of antirestriction genes on conjugative plasmids

Interaction between the F plasmid TraA (F‐pilin) and TraQ proteins

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

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