the gram - positive pathogen staphylococcus aureus is a significant cause of morbidity and mortality worldwide . the success of this pathogen can be partially attributed to its remarkable ability to infect nearly every vertebrate organ . several virulence traits endow s.  aureus with the capacity to manipulate and survive within the host environment . these include , but are not limited to , the production of hemolysins , the secretion of proteases , and the synthesis of the organism s clinically distinguishing golden pigment , staphyloxanthin ( 1 ) . treatment of s.  aureus infections are confounded by the incidence of antibiotic resistance strains . in fact , methicillin - resistant s.  aureus ( mrsa ) is now the predominant cause of staphylococcal infection in the united states ( 2 ) , and mortality due to mrsa infection in the united states is greater than that of aids and tuberculosis combined ( 2 , 3 ) . once thought to be confined to the clinical setting , mrsa has been increasingly isolated from community - acquired infections ( 4 ) . these facts highlight the need to understand the physiology and virulence strategies of this organism in an effort to uncover novel therapeutic targets for the treatment of mrsa . therefore , a library of defined genetic mrsa mutants would represent a valuable resource for the community of researchers focused on understanding the pathogenesis of s.  aureus infections . constructing mutants of s.  aureus in a laboratory involves , primarily , random - transposition and allelic - replacement strategies , which can be tedious and cause secondary mutations that confound results ( 5 ) . a defined transposon library was previously created with the methicillin - sensitive strain s.  aureus newman , enabling a number of exciting discoveries ( 69 ) . this resource made clear the value that an openly accessible , defined transposon library could have on the staphylococcal research community . fey et al . have taken on this challenge by creating a defined transposon library of s.  aureus that is distributed through the network on antimicrobial resistance in staphylococcus aureus ( narsa ) ( 10 ) . this library was constructed with the mrsa strain je2 , a usa300 derivative which represents the predominant clonal lineage of mrsa isolated from infected patients in the united states ( 11 ) . a mariner - based bursa transposon mutagenesis approach was used to create random insertion mutations throughout the genome ( 6 ) . erythromycin resistance was used to select for strains containing a transposon insertion , and the sites of mutation were then mapped in order to determine the site of insertion . strains containing a transposon insertion within the first 90% of a coding sequence ( cds ) were prioritized and included in the nebraska transposon mutant library ( ntml ) . this selection resulted in the final collection of the 1,952 mutants that comprise the ntml , representing 76% coverage of the predicted cdss within the je2 genome . interestingly , 579 putative cdss were not identified in the screening process for the ntml , raising the intriguing possibility that these represent the suite of essential genes within s.  aureus which could include novel targets for therapeutic intervention . as a demonstration of the utility and comprehensiveness of the ntml , fey et al . screened for mutants altered in four phenotypes that clinically distinguish s.  aureus from other bacteria : the production of hemolysin , the ability to produce the golden pigment staphyloxanthin , the production of proteases , and the ability to utilize mannitol as the sole carbon source . the power of the nmtl is indicated by the fact that the authors identified mutants known to be impaired for these respective pathways . these mutants include the alpha hemolysin involved in lysing red blood cells , the known staphyloxanthin biosynthesis genes , two secreted proteases , and all of the genes in the mannitol utilization operons ( 1215 ) . in total , 71 mutants with altered hemolytic activity , 62 mutants with altered protease production , and 38 mutants affected for pigment production were identified . some of these mutants have been identified in previous screens , while others have not been associated with these processes ( 7 , 1620 ) . these findings further refine our knowledge of the genetic networks involved in these pathways and underscore the effectiveness of using the ntml in a phenotypic screening approach . individual mutants or the entire ntml can be ordered free of charge by members of the network on antimicrobial resistance in staphylococcus aureus ( narsa ) . a list of the mutants can be found at the following url : http://app1.unmc.edu / fgx/. fey et al . are also in the process of making additional tools available for the narsa community , including codon - optimized transcription reporters that can be utilized in conjunction with the ntml for further study . thus far , 2,588 individual mutants have been sent to independent investigators , and 20 laboratories throughout the world have received the entire ntml , highlighting the demand for this valuable resource ( personal communication ) . due to the fact that the ntml was created using a protocol that required changing incubation temperatures , a procedure known to cause off - target mutations in major s.  aureus virulence two - component systems ( 9 ) , researchers should be cautious when interpreting results from a phenotypic screen using the ntml . in keeping with this , investigators should validate any observed phenotypes of interest by backcrossing insertions into clean strain backgrounds , inactivating associated genes through traditional allelic - replacement strategies , and/or complementing phenotypes of interest by providing a full - length copy of the gene in trans . as the narsa community begins to make use of the powerful genetic tools provided by fey and his collaborators , research into s.  aureus
abstractin  a genetic resource for rapid and comprehensive phenotype screening of nonessential staphylococcus aureus genes  ( mbio 4(2):e00537 - 12 , doi : 10.1128/mbio.00537 - 12 , 2013 ) , fey et al . 
 describe the creation and application of a defined transposon mutant library of methicillin - resistant s.  aureus . 
 this library is well organized and made accessible to the research community through an easily navigable central repository . 
 the mutant library promises to be a significant resource for researchers seeking a greater understanding of this pathogen .