References

Project:

Staphylococcus aureus N315

About this genome

*Whole genome sequencing of meticillin-resistant Staphylococcus aureus.*
Kuroda M.,Ohta T.,Uchiyama I.,Baba T.,Yuzawa H.,Kobayashi I.,Cui L.,Oguchi A.,Aoki K.,Nagai Y.,Lian J.,Ito T.,Kanamori M.,Matsumaru H.,Maruyama A.,Murakami H.,Hosoyama A.,Mizutani-Ui Y.,Takahashi NK.,Sawano T.,Inoue R.,Kaito C.,Sekimizu K.,Hirakawa H.,Kuhara S.,Goto S.,Yabuzaki J.,Kanehisa M.,Yamashita A.,Oshima K.,Furuya K.,Yoshino C.,Shiba T.,Hattori M.,Ogasawara N.,Hayashi H.,Hiramatsu K. Lancet 357 (2001) 1225-40 [PMID: 11418146] Abstract
Abstract: BACKGROUND: Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS: Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS: The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION: The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Whole genome sequencing of meticillin-resistant Staphylococcus aureus.

Kuroda M.,Ohta T.,Uchiyama I.,Baba T.,Yuzawa H.,Kobayashi I.,Cui L.,Oguchi A.,Aoki K.,Nagai Y.,Lian J.,Ito T.,Kanamori M.,Matsumaru H.,Maruyama A.,Murakami H.,Hosoyama A.,Mizutani-Ui Y.,Takahashi NK.,Sawano T.,Inoue R.,Kaito C.,Sekimizu K.,Hirakawa H.,Kuhara S.,Goto S.,Yabuzaki J.,Kanehisa M.,Yamashita A.,Oshima K.,Furuya K.,Yoshino C.,Shiba T.,Hattori M.,Ogasawara N.,Hayashi H.,Hiramatsu K.
Lancet 357 (2001) 1225-40 [PMID: 11418146] Abstract

Abstract: BACKGROUND: Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS: Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS: The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION: The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Journal

*Requirement of Staphylococcus aureus* ATP-binding cassette-ATPase FhuC for iron-restricted growth and evidence that it functions with more than one iron transporter.**
Speziali CD.,Dale SE.,Henderson JA.,Vinés ED.,Heinrichs DE. J. Bacteriol. 188 (2006) 2048-55 [PMID: 16513734] Abstract
Abstract: In Staphylococcus aureus, fhuCBG encodes an ATP-binding cassette (ABC) transporter that is required for the transport of iron(III)-hydroxamates; mutation of either fhuB or fhuG eliminates transport. In this paper, we describe construction and characterization of an S. aureus fhuCBG deletion strain. The delta fhuCBG::ermC mutation not only resulted in a strain that was incapable of growth on iron(III)-hydroxamates as a sole source of iron but also resulted in a strain which had a profound growth defect in iron-restricted laboratory media. The growth defect was not a result of the inability to transport iron(III)-hydroxamates since S. aureus fhuG::Tn917 and S. aureus fhuD1::Km fhuD2::Tet mutants, which are also unable to transport iron(III)-hydroxamates, do not have similar iron-restricted growth defects. Complementation experiments demonstrated that the growth defect of the delta fhuCBG::ermC mutant was the result of the inability to express FhuC and that this was the result of an inability to transport iron complexed to the S. aureus siderophore staphylobactin. Transport of iron(III)-staphylobactin is dependent upon SirA (binding protein), SirB (permease), and SirC (permease). S. aureus expressing FhuC with a Walker A K42N mutation could not utilize iron(III)-hydroxamates or iron(III)-staphylobactin as a sole source of iron, supporting the conclusion that FhuC, as expected, functions with FhuB, FhuG, and FhuD1 or FhuD2 to transport iron(III)-hydroxamates and is the "genetically unlinked" ABC-ATPase that functions with SirA, SirB, and SirC to transport iron(III)-staphylobactin. Finally, we demonstrated that the delta fhuCBG::ermC strain had decreased virulence in a murine kidney abscess model.
*Nucleotide substitutions in Staphylococcus aureus* strains, Mu50, Mu3, and N315.**
Ohta T.,Hirakawa H.,Morikawa K.,Maruyama A.,Inose Y.,Yamashita A.,Oshima K.,Kuroda M.,Hattori M.,Hiramatsu K.,Kuhara S.,Hayashi H. DNA Res. 11 (2004) 51-6 [PMID: 15141945] Abstract
Abstract: A specific phenotype of Staphylococcus aureus strains Mu50 and Mu3 is characterized by thickened cell wall and moderate resistance to vancomycin. The N315 strain is a prototype of methicillin-resistant S. aureus (MRSA), but it is methicillin susceptible, despite carrying the mecA resistance gene. Here, we revised differences in the sequences of Mu50 and N315, referencing that of Mu3 which were assumed to be of one lineage. The 362 ORFs diverse between Mu50 and N315 were picked up, and the corresponding ones in three strains were re-sequenced. This defined 213 ORFs diverse between Mu50 and N315, and 9 between Mu50 and Mu3. The fixed diversities of 174 ORFs (except for 39 silent ORFs from 213), including nucleotide substitution (NSs), frame shift, and truncation were grouped into three major functional categories, which were transport (14.9% in the 174 diverse ORFs), metabolism of carbohydrates (5.7%), and RNA synthesis (9.6%). The other gene categories had small diversities. These gene categories seemed to be functionally decisive for the Mu50-specific characters, the thickened cell wall and moderate vancomycin resistance. All of the diverse genes and the high quality sequence of Mu50 can be viewed at the web site (http://133.5.48.239/VRSA/).
*Role of siderophore biosynthesis in virulence of Staphylococcus aureus: identification and characterization of genes involved in production of a siderophore.*
Dale SE.,Doherty-Kirby A.,Lajoie G.,Heinrichs DE. Infect. Immun. 72 (2004) 29-37 [PMID: 14688077] Abstract
Abstract: Molecular determinants underlying the production of siderophores in the human and animal pathogen Staphylococcus aureus and the contribution of siderophore production to the virulence of this bacterium have, until now, remained undefined. Here, we show that S. aureus strains RN6390 and Newman produce siderophore when the cells are starved for iron. We further identified and characterized a nine-gene, iron-regulated operon, designated sbn and situated between sirABC and galE on the S. aureus chromosome, that is involved in the production of a siderophore. Mutation of the sbnE gene, in both RN6390 and Newman, eliminates the ability of these strains to produce a siderophore under iron-limited growth conditions, while introduction of multicopy sbnE into sbnE mutants complemented the inability of the mutants to produce the siderophore. sbnE mutants, in both the RN6390 and Newman backgrounds, displayed a drastic growth deficiency, compared to the wild type, in iron-restricted growth medium, whereas no such deficiency was observed during growth in iron-replete medium. Complemented mutants showed a restored ability to grow under iron restriction. We further showed that an sbnE mutant was compromised in a murine kidney abscess model of S. aureus infection, illustrating the importance of siderophore production to the pathogenicity of S. aureus. sbn genes were present in all S. aureus strains tested (and all S. aureus genome sequences) but were undetectable in any of the 13 coagulase-negative staphylococci tested, including Staphylococcus epidermidis.
*Identification and characterization of fhuD1 and fhuD2, two genes involved in iron-hydroxamate uptake in Staphylococcus aureus.*
Sebulsky MT.,Heinrichs DE. J. Bacteriol. 183 (2001) 4994-5000 [PMID: 11489851] Abstract
Abstract: Staphylococcus aureus can utilize several hydroxamate siderophores for growth under iron-restricted conditions. Previous findings have shown that S. aureus possesses a cytoplasmic membrane-associated traffic ATPase that is involved in the specific transport of iron(III)-hydroxamate complexes. In this study, we have identified two additional genes, termed fhuD1 and fhuD2, whose products are involved in this transport process in S. aureus. We have shown that fhuD2 codes for a posttranslationally modified lipoprotein that is anchored in the cytoplasmic membrane, while the deduced amino acid sequence predicts the same for fhuD1. The predicted FhuD1 and FhuD2 proteins share 41.0% identity and 56.4% total similarity with each other, 45.9 and 49.1% total similarity with the FhuD homolog in Bacillus subtilis, and 29.3 and 24.6% total similarity with the periplasmic FhuD protein from Escherichia coli. Insertional inactivation and gene replacement of both genes showed that while FhuD2 is involved in the transport of iron(III) in complex with ferrichrome, ferrioxamine B, aerobactin, and coprogen, FhuD1 shows a more limited substrate range, capable of only iron(III)-ferrichrome and iron(III)-ferrioxamine B transport in S. aureus. Nucleotide sequences present upstream of both fhuD1 and fhuD2 predict the presence of consensus Fur binding sequences. In agreement, transcription of both genes was negatively regulated by exogenous iron levels through the activity of the S. aureus Fur protein.
*Identification and characterization of a membrane permease involved in iron-hydroxamate transport in Staphylococcus aureus.*
Sebulsky MT.,Hohnstein D.,Hunter MD.,Heinrichs DE. J. Bacteriol. 182 (2000) 4394-400 [PMID: 10913070] Abstract
Abstract: Staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine. An S. aureus mutant defective in the ability to transport ferric hydroxamate complexes was isolated from a Tn917-LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. Chromosomal DNA flanking the Tn917-LTV1 insertion was identified by sequencing of chromosomal DNA isolated from the mutant. This information localized the transposon insertion to a gene whose predicted product shares significant similarity with FhuG of Bacillus subtilis. DNA sequence information was then used to clone a larger fragment of DNA surrounding the fhuG gene, and this resulted in the identification of an operon of three genes, fhuCBG, all of which show significant similarities to ferric hydroxamate uptake (fhu) genes in B. subtilis. FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while FhuC shares significant homology with ATP-binding proteins. Given this, the S. aureus FhuCBG proteins were predicted to be part of a binding protein-dependent transport system for ferric hydroxamates. Exogenous iron levels were shown to regulate ferric hydroxamate uptake in S. aureus. This regulation is attributable to Fur in S. aureus because a strain containing an insertionally inactivated fur gene showed maximal levels of ferric hydroxamate uptake even when the cells were grown under iron-replete conditions. By using the Fur titration assay, it was shown that the Fur box sequences upstream of fhuCBG are recognized by the Escherichia coli Fur protein.