Prevalence of Agr phase variants in Staphylococcus aureus

Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing Accessory Gene Regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr negative strains and screening for phenotypic reversion of haemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative MRSA isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. To assess the significance of our findings we screened a series of primary clinical isolates, which had undergone minimal handling post-isolation, and successfully identified a fraction which were Agr phase variants. Taken together, we propose a model where some Agr-negative S. aureus strains are phase variants who can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress.

Effectiveness of Novel Ankle Prophylactic Compared With Lace-Up Brace or Tape

Context: Conventional ankle prophylactics restrict harmful ankle inversion motions that lead to injury. But these existing prophylactics also limit other ankle motions, potentially leading to detriments in functional joint capacity. The ankle roll guard (ARG) may alleviate the prevailing issues of existing ankle prophylactics and prevent harmful ankle inversion, while allowing other joint motions. Objective: This technical report sought to compare the ARG’s ability to prevent ankle inversion, but not restrict other ankle motions with existing prophylactics. Design: Repeated-measures study. Setting: Motion capture laboratory. Participants: Thirty participants. Intervention: Each participant had dominant limb ankle kinematics recorded during 5 successful trials of a sudden inversion event and 30-cm drop landing task with each of 4 conditions (ARG, ASO ankle stabilizer [brace], closed-basket weave athletic tape [tape], and unbraced [control]). Main Outcome Measures: Peak ankle inversion angle, range of inversion motion (ROM), and time to peak inversion during the sudden inversion event, and ankle plantar- and dorsiflexion ROM during the drop landing were submitted to a 1-way repeated-measures analysis of variance to test the main effect of prophylaxis. Results: Participants exhibited greater inversion ROM with control compared with tape (P = .001), and greater plantar- and dorsiflexion ROM with ARG and control compared with brace (P = .02, P = .001) and tape (P = .02, P < .001). It took significantly longer to reach peak ankle inversion with brace and tape compared with ARG (P < .001, P = .001) and control (P = .01, P = .01). No significant difference in peak ankle inversion was observed between any condition (P > .05). Conclusion: The ARG may prevent ankle inversion angles where injury is thought to occur (reportedly >41°), but is less restrictive than existing prophylactics. The less restrictive ARG may make its use ideal during rehabilitation as it allows ankle plantar- and dorsiflexion motions, while preventing inversion related to injury.

Multiple large inversions and breakpoint rewiring of gene expression in the evolution of the fire ant social supergene

Supergenes consist of co-adapted loci that segregate together and are associated with adaptive traits. In the fire ant Solenopsis invicta , two ‘social’ supergene variants regulate differences in colony queen number and other traits. Suppressed recombination in this system is maintained, in part, by a greater than 9 Mb inversion, but the supergene is larger. Has the supergene in S. invicta undergone multiple large inversions? The initial gene content of the inverted allele of a supergene would be the same as that of the wild-type allele. So, how did the inversion increase in frequency? To address these questions, we cloned one extreme breakpoint in the fire ant supergene. In doing so, we found a second large (greater than 800 Kb) rearrangement. Furthermore, we determined the temporal order of the two big inversions based on the translocation pattern of a third small fragment. Because the S. invicta supergene lacks evolutionary strata, our finding of multiple inversions may support an introgression model of the supergene. Finally, we showed that one of the inversions swapped the promoter of a breakpoint-adjacent gene, which might have conferred a selective advantage relative to the non-inverted allele. Our findings provide a rare example of gene alterations arising directly from an inversion event.

Development of molecular markers and linkage maps for the Carthamus species C. tinctorius and C. oxyacanthus

A set of SSR and RFLP markers for safflower ( Carthamus tinctorius ) and jeweled distaff thistle ( C. oxyacanthus ) was generated from cDNA and genomic libraries and by mining public and proprietary sequence databases. In total, 1412 PCR-based markers and 75 RFLP markers were screened and polymorphic loci were mapped in an intraspecific F2 population of C. tinctorius and an interspecific BC1 population of C. tinctorius × C. oxyacanthus. The two populations shared one common parent and the resulting linkage maps could be compared for synteny. The level of polymorphism was low in both populations and only 8.2% and 13.7% of the analyzed markers could be mapped in the intraspecific and interspecific maps, respectively. The two maps showed significant colinearity of markers in several regions and an apparent translocation or inversion event on one linkage group. Noticeable segregation distortion was found on one linkage group of the C. tinctorius map and dense clustering of loci occurred on several linkage groups of the C. oxyacanthus map. The two maps represent the first major linkage analysis of Carthamus species. The molecular tools will be useful for a variety of genetic and genomic applications in safflower and its related species and have been used in our laboratory to map a flower color gene in C. tinctorius.

DNA Supercoiling and the Lrp Protein Determine the Directionality of fim Switch DNA Inversion in Escherichia coli K-12

ABSTRACT Site-specific recombinases of the integrase family usually require cofactors to impart directionality in the recombination reactions that they catalyze. The FimB integrase inverts the Escherichia coli fim switch (fimS) in the on-to-off and off-to-on directions with approximately equal efficiency. Inhibiting DNA gyrase with novobiocin caused inversion to become biased in the off-to-on direction. This directionality was not due to differential DNA topological distortion of fimS in the on and off phases by the activity of its resident P fimA promoter. Instead, the leucine-responsive regulatory (Lrp) protein was found to determine switching outcomes. Knocking out the lrp gene or abolishing Lrp binding sites 1 and 2 within fimS completely reversed the response of the switch to DNA relaxation. Inactivation of either Lrp site alone resulted in mild on-to-off bias, showing that they act together to influence the response of the switch to changes in DNA supercoiling. Thus, Lrp is not merely an architectural element organizing the fim invertasome, it collaborates with DNA supercoiling to determine the directionality of the DNA inversion event.