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JOURNAL OF BACTERIOLOGY, May 1985, p. 584-590 0021-9193/85/050584-07$02.00/0 Copyright X 1985, American Society for Microbiology

Vol. 162, No. 2

DNA Transfer Occurs During a Cell Surface Contact Stage of F Sex Factor-Mediated Bacterial Conjugation

Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213Received 17 September 1984/Accepted 4 February 1985

Donor bacteria containing JCFL39, a temperature-sensitive traD mutant of the F sex factor, were used at the nonpermissive temperature to accumulate stable mating pairs with recipient cells. At this stage in conjugation, extracellular F pili were removed by treatment with 0.01% sodium dodecyl sulfate. Upon then shifting to the permissivetemperature for JCFL39, transfer of the F plasmid was observed. The mating pairs that were accumulated with JCFL39 at the nonpermissive temperature were readily observed by electron microscopy in wall-to-wall contact with the recipient bacteria. These results demonstrate that the traD product, which is known to be required in transferring DNA to a recipient bacterium, acts after the stage at whichextracellular F pili are required. In addition, we concluded that DNA transfer takes place while donor and recipient cells are in surface contact and not necessarily through an extended F pilus as envisioned in some models of bacterial

Conjugation is the process whereby DNA is transferred from a donor to a recipient bacterium by a mechanism that involves contact between the cells.Most conjugation studies have been performed with gram-negative bacteria and in particular have centered on Escherichia coli and its sex factor, F. A central feature of F-mediated conjugation is the function of the F pilus, a hairlike extracellular filament that is produced in one or few copies by an F plasmid-containing donor bacterium (9). Although there is strong evidence that the F pilus isessential for the formation of the initial contact between a donor and a recipient bacterium (4, 7), there is still a degree of uncertainity as to the role that this organelle plays in conjugative DNA transfer. The earliest observations (5, 16) indicated the possibility of direct cell surface contact between conjugating bacteria, but since these studies predated the discovery of F pili, no criticalexperiments were performed at that time to distinguish between possible roles for the sex pilus. Brinton's studies on F pili led him to propose a class of models in which the F pilus is directly involved in conjugative DNA transfer (6). However, no direct evidence that demonstrates an association between F pili and DNA that is being transferred conjugatively has been reported in the literature. Asan alternative, Curtiss (10) and Marvin and Hohn (18) have suggested that F pili might function by retracting and thereby drawing the donor and recipient cell surfaces together, at which point DNA transfer would occur. This idea is central to the currently favored model for conjugative transfer by F-like plasmids. The central features of this model have most recently been reviewed by Willetts andSkurray (26) and are presented in Fig. 1. The model envisions conjugation as proceeding through a series of ordered stages of cell surface and DNA metabolism events. Much of the evidence for this model is based upon the phenotypes of F plasmid mutants that are deficient in transfer (tra) (26). Mutants in traA,L,E,K,B,V,W,C,U, F,H, or the first part of traG do not synthesize F pili and are defectivein all stages of conjugation. Mutants in traN and the second part of traG synthesize F pili and make unstable, but not stable (shear-resistant), cell surface contacts (17).

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Recipient bacteria which lack the outer membrane ompA protein are also unable to form stable mating pairs (21). Mutants in traM,D,I,Z, (and probably tra 1) are piliated and are able to...
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