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Running is better than weight training at reversing signs of ageing. One possibility is that a change in metabolite composition affects gene transcription, but since aconitase has been reported to be inactive during log-phase growth 40 , it is hard to envision that this mechanism is in play. We therefore looked for other possible functions for this protein.
It has been reported in other bacteria that when the iron-sulfur network is lost during iron limitation or oxidative stress, aconitase functions as a pleiotropic posttranscriptional regulator by binding to specific mRNA transcripts and regulating their translation 41 , Our in vitro growth experiments were not done under iron deprivation, but in preliminary in vitro experiments, iron limitation did not alter the growth phenotype or increase the in vitro fitness of FA19 penA41 acnB GD.
However, it is likely that bacteria are exposed to iron-limiting conditions during infections of the host. Even if aconitase acts as a posttranslational regulator of protein translation, however, that still does not explain such a large difference in gene transcription. While it is clear that the acnB GD allele increases fitness, it was not sufficient to bring the level of fitness to that of the compensatory mutant LV41C. We therefore tested whether an additional mutation found in LV41C, a frameshift mutation in the phase-variable pglG gene that restores the complete open reading frame, would increase fitness but found no evidence that this mutation alters fitness relative to LV41C.
There remain additional untested mutations in the noncoding region downstream of ftsY , and it is possible that these mutations alter transcription or transcript stability of the associated gene.
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This scenario may also explain the fitness advantage of LV41E, which did not contain any mutations in protein-encoding regions. In summary, here we have demonstrated that two clinically relevant mosaic penA alleles, which result in Cro resistance in N.
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In vivo , compensatory mutations were readily selected in a Cro r strain carrying the penA41 allele that abrogated the fitness disadvantage but not for the more attenuating penA89 allele. Differences in vivo and in vitro for the acnB GD mutation suggest that AcnB may have a yet-undiscovered function s that leads to the fitness advantage observed in vivo.
Ongoing research in this area should lead to novel glimpses into gonococcal metabolism and physiology. Our findings also suggest that ESC resistance may not spread rapidly but that compensatory evolution may allow some ESC r strains to be maintained in communities and potentially spread. Continued surveillance for ESC r strains will be essential to identify whether the compensatory mutations that we have identified in a surrogate infection model are present in ESC r isolates from natural infections. The antibiotic-susceptible laboratory strain FA19 52 was used as the background strain for all fitness studies.
Streptomycin resistance is required for mouse infection studies 34 ; therefore, the rpsL1 gene from FA, which encodes a mutant form of ribosomal protein S12 that confers streptomycin resistance 53 , was transformed into strain FA19 by allelic exchange All FA19 strain variants used in this study harbored the rpsL1 allele. Strain FA19 penA41 cat was constructed by introducing the ClaI fragment carrying a chloramphenicol acetyltransferase cat gene from the Neisseria chromosomal integration vector pGCC5 55 into a nonessential intergenic region in FA19 penA Passage from frozen stocks was minimized to reduce the risk of acquiring secondary mutations.
For competitive infections and cocultures, GCB agar plates containing antibiotic selection were used to isolate the mutants as follows: ceftriaxone, 0. Differences in growth rate were measured by comparing the average numbers of minutes that it took to reach an OD of 0. The MICs of Cro, penicillin G, erythromycin, ciprofloxacin, and chloramphenicol were determined by agar dilution as described previously Competitive coculture was used to compare the fitness of Cro s or Cro r parent strains with those of mutant strains FA19 penA41 and FA19 penA89 or the in vivo -selected compensatory mutants of FA19 penA Experiments were conducted three times, and the geometric mean CI at each time point was determined.
E; Sigma-Aldrich, St. E; Sigma-Aldrich suspended in sesame oil when competing compensatory mutant strains against parent and wild-type strains. Mice were also given streptomycin sulfate, vancomycin, and trimethoprim to reduce the overgrowth of commensal flora as described previously A CI was determined whereby the ratio of mutant to wild-type bacteria in vaginal cultures at each individual time point was divided by the ratio of mutant to wild-type bacteria in the inoculum.
Experiments were conducted two or three times for each mixture. A mate-pair library was constructed using Nextera XT standard kits Illumina FC and FC and prepared for emulsion-based amplification and paired-end sequencing. Each read was bases with two reads per insert plus a barcode. No deletions larger than 3 nucleotides e. Primers were made to these regions, and DNA surrounding the low-coverage regions was amplified by PCR and sequenced by Sanger sequencing.
In all cases, the sequences were identical to FA Following expression in E. The slopes of each reaction within the first minute were used to calculate the initial velocity of the enzymes. The appropriate strains were grown in liquid GC broth medium with glucose as the main carbon source. RNA reference samples were diluted , using RNase-free water. The membrane was incubated in PBS-Tween containing 2. PilM was used as an internal loading control because its expression does not appear to change in different phases of growth; however, it was not used to normalize protein expression between strains because its expression appears to vary between strains.
Run parameters and data were collected using Illumina BaseSpace. To determine differentially expressed genes, the differential expression for RNA-seq command was run. We thank Cara Olsen for help with statistical analyses and Ian McDonald for helping with the aconitase assays. We also thank William Shafer at Emory University for helpful discussions and critical review of the manuscript. The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the Uniformed Services University of the Health Sciences, the Department of Defense, or the University of North Carolina.
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
In vivo -selected compensatory mutations restore the fitness cost of mosaic penA alleles that confer ceftriaxone resistance in Neisseria gonorrhoeae. National Center for Biotechnology Information , U. Journal List mBio v. Published online Apr 3.