To enhance the research capabilities of investigators interested in Staphylococcus aureus, the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) isolate USA300. To demonstrate the utility of this library for large-scale screening of phenotypic alterations, we spotted the library on indicator plates to assess hemolytic potential, protease production, pigmentation, and mannitol utilization. As expected, we identified many genes known to function in these processes, thus validating the utility of this approach. Importantly, we also identified genes not previously associated with these phenotypes. In total, 71 mutants displayed differential hemolysis activities, the majority of which were not previously known to influence hemolysin production. Furthermore, 62 mutants were defective in protease activity, with only 14 previously demonstrated to be involved in the production of extracellular proteases. In addition, 38 mutations affected pigment formation, while only 7 influenced mannitol fermentation, underscoring the sensitivity of this approach to identify rare phenotypes. Finally, 579 open reading frames were not interrupted by a transposon, thus providing potentially new essential gene targets for subsequent antibacterial discovery. Overall, the Nebraska Transposon Mutant Library represents a valuable new resource for the research community that should greatly enhance investigations of this important human pathogen.
(http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. H. influenzae core-and supra-genome characterization The genomes of 9 non-typeable
The distributed-genome hypothesis (DGH) states that pathogenic bacteria possess a supragenome that is much larger than the genome of any single bacterium and that these pathogens utilize genetic recombination and a large, noncore set of genes as a means of diversity generation. We sequenced the genomes of eight nasopharyngeal strains of Streptococcus pneumoniae isolated from pediatric patients with upper respiratory symptoms and performed quantitative genomic analyses among these and nine publicly available pneumococcal strains. Coding sequences from all strains were grouped into 3,170 orthologous gene clusters, of which 1,454 (46%) were conserved among all 17 strains. The majority of the gene clusters, 1,716 (54%), were not found in all strains. Genic differences per strain pair ranged from 35 to 629 orthologous clusters, with each strain's genome containing between 21 and 32% noncore genes. The distribution of the orthologous clusters per genome for the 17 strains was entered into the finite-supragenome model, which predicted that (i) the S. pneumoniae supragenome contains more than 5,000 orthologous clusters and (ii) 99% of the orthologous clusters (ϳ3,000) that are represented in the S. pneumoniae population at frequencies of >0.1 can be identified if 33 representative genomes are sequenced. These extensive genic diversity data support the DGH and provide a basis for understanding the great differences in clinical phenotype associated with various pneumococcal strains. When these findings are taken together with previous studies that demonstrated the presence of a supragenome for Streptococcus agalactiae and Haemophilus influenzae, it appears that the possession of a distributed genome is a common host interaction strategy.Streptococcus pneumoniae is a gram-positive bacterium commonly found in the nasopharynges of healthy persons, predominantly children. In addition to its commensal form, S. pneumoniae is also a major cause of morbidity and mortality worldwide. S. pneumoniae infection can cause meningitis and bacteremia, as well as many mucosal diseases such as pneumonia, sinusitis, and otitis media (OM). Worldwide, S. pneumoniae infections are estimated to result in 1.1 million deaths a year, predominantly from pneumonia, and even in the United States pneumococcus disease is one of the top 10 causes of death (21). The economic burden associated with S. pneumoniae infections is tremendous, because it is the causative agent for 30 to 50% of OM infections worldwide, and in the United States alone the cost of OM, which is the most prevalent infectious disease among children, is estimated at $5 billion annually (5, 12).S. pneumoniae played a critical role in the demonstration that DNA is the hereditary genetic material. In 1944 Avery and colleagues showed that DNA is the transforming factor identified by Griffith as being capable of making avirulent S. pneumoniae lethal (1). Since then, S. pneumoniae has served as a model organism for the study of bacterial transformation. It contains an inducible system for the...
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