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PNAS 102 (43): 15617-15622

Copyright © 2005 by the National Academy of Sciences.

A paracrine peptide sex pheromone also acts as an autocrine signal to induce plasmid transfer and virulence factor expression in vivo

Josephine R. Chandler *, Helmut Hirt *, {dagger}, and Gary M. Dunny *, {ddagger}

*Department of Microbiology, University of Minnesota, MMC 196, 420 Delaware Street SE, Minneapolis, MN 55455; and {dagger}Division of Biology, Kansas State University, 232 Ackert Hall, Manhattan, KS 66506



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Fig. 1.. Model of regulation of pheromone and pheromone response in a donor cell. (A) Donors in pure culture. PrgY reduces endogenous cCF10, and iCF10 neutralizes remaining cCF10. PrgX represses Asc10 expression so that Asc10 is not induced. (B) Donors induced by cCF10 from a nearby recipient cell (i) or growth in plasma (ii). In both cases, PrgX repression is abolished by binding of imported cCF10 and Asc10 is expressed. Induction in plasma (ii) occurs through two possible mechanisms: a small host molecule (probably a peptide) with cCF10 activity acts as a direct signal to induce Asc10 expression, or a host factor (possibly albumin; see text) sequesters iCF10, altering the ratio of peptides and allowing endogenous cCF10 to act as an autocrine inducer.

 


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Fig. 2.. Mutations in the ccfA gene, which encodes cCF10 pheromone (JRC101 and JRC102), decrease pCF10 recipient ability compared with wild type (CK104). OG1SSp(pCF10) was used as a donor strain. Overnight cultures were diluted 1:10 in THB and were grown for 1 hr before donors and recipients were combined to begin mating. Mating was allowed to proceed for the time indicated before plating. (A) Plasmid transfer depicted as transconjugants per donor cell. Experiments were done in duplicate; error bars represent one standard deviation of the mean. (B) Total numbers of transconjugants per ml.

 


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Fig. 3.. Endogenous pheromone is required for induction by plasma. (A) pCF10 transfer and Asc10 expression of donor strains JRC102(pCF10) or CK104(pCF10). pCF10 transfer and Asc10 expression are induced by plasma in strain CK104 (wild-type) but not in the cCF10-negative strain JRC102. Strain OG1Sp was used as a recipient for mating. For quantification of plasmid transfer, overnight cultures of recipients and donors were diluted 1:10 in M9 medium (with or without 1 ng/ml cCF10) or in plasma and grown for 2 hr before donors and recipients were combined to begin mating. Mating mixtures were incubated for 10 min before plating. Plasmid transfer is depicted as transconjugants per donor. Experiments were done in duplicate; error bars represent 1 SD about the mean. Immunoblot analysis for Asc10 by using a polyclonal anti-Asc10 antibody is depicted below the mating results; samples are in the same order. As for the mating assay, overnight cultures were diluted 1:10 in M9 medium (with or without 1 ng/ml cCF10) or in plasma. Cell extracts were washed twice before harvesting. An equivalent amount of protein was spotted for each sample. (B) Retention of albumin by fractionation of plasma on an iCF10 affinity column. The iCF10 peptide was coupled with the carboxyl terminus to Sepharose 6B. The matrix material was incubated with human plasma and eluted with acetonitrile (see Supporting Text, which is published as supporting information on the PNAS web site). Eluate was separated on an SDS/7.5% PAGE gel and silver stained. Lane 1, human plasma 1:1000; lane 2, acetonitrile eluate from the affinity column; lane 3, purified (commercially obtained) human serum albumin (100 ng).

 


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Fig. 4.. Regulation of plasmid transfer and Qs (encoding iCF10) expression by PrgY and by endogenous pheromone. (A) Transfer of pCF10 and pCF389 (prgY-null) out of CK104 and JRC102 donor strains. OG1Sp was used as a recipient strain. Pheromone induction (0 and 1 ng/ml cCF10 samples) experiments were carried out by a protocol different from that with the longer mating time (30-min sample), with the data displayed side-by-side for comparison. For 0 and 1 ng/ml cCF10, donors and recipients were grown overnight in M9 medium and diluted 1:10, then grown an additional 2 hr in the presence or absence of 1 ng/ml cCF10 and combined for a 10-min mating before plating. For the 30-min samples, donor and recipient strains were grown overnight in THB, diluted 1:10, and grown for 1 hr in the absence of cCF10, then they were combined for 30 min to allow induction of donors by recipient pheromone before plating. Plasmid transfer is depicted as transconjugants per donor. Experiments were done in duplicate; error bars represent one SD about the mean. Clumping assay results for each strain are shown below. Strains induced for plasmid transfer express Asc10 and clump in liquid culture (see text), which causes uneven settling at the bottom of the well (lane 4) of a 96-well plate. Overnight cultures were grown in THB without pheromone and were shaken for 2 hr before a picture was taken. (B) Location of the Qs probe within the regulatory region of pCF10 and pCF389. The digoxigenin-labeled RNA probes were generated by in vitro transcription. The 3' end of the probes is indicated by an arrowhead. (C) Results of Northern blotting analysis of Qs RNA levels. For Northern analysis, 1 µg of total RNA was separated on a 2% agarose gel, transferred onto nylon membrane, and hybridized with the Qs probe shown in B. The arrow indicates Qs. Strains depicted are as follows: JRC102 (lanes 1 and 3) and CK104 (lanes 2 and 4). These strains harbor the plasmid pCF10 (lanes 1 and 2) or pCF389 (lanes 3 and 4). Relative amounts of Qs as determined by densitometry are displayed below each lane. These data were obtained by determining the profile area with the Bio-Rad MOLECULAR ANALYST (Version 21) software.

 


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