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PubMed1995

Ciliate telomerase RNA structural features.

Telomerase RNA is an integral part of telomerase, the ribonucleoprotein enzyme that catalyzes the synthesis of telomeric DNA. The RNA moiety contains a templating domain that directs the synthesis of a species-specific telomeric repeat and may also be important for enzyme structure and/or catalysis. Phylogenetic comparisons of telomerase RNA sequences from various Tetrahymena spp. and hypotrich ciliates have revealed two conserved secondary structure models that share many features. We have cloned and sequenced the telomerase RNA genes from an additional six Tetrahymena spp. (T. vorax, T. borealis, T. australis, T. silvana, T. capricornis and T. paravorax). Inclusion of these sequences, most notably that from T. paravorax, in a phylogenetic comparative analysis allowed us to more narrowly define structural elements that may be necessary for a minimal telomerase RNA. A primary sequence element, positioned 5' of the template and conserved between all previously known ciliate telomerase RNAs, has been reduced from 5'-(C)UGUCA-3' to the 4 nt sequence 5'-GUCA-3'. Conserved secondary structural features and the impact they have on the general organization of ciliate telomerase RNAs is discussed.

Animals↗

PubMed2002

A critical stem-loop structure in the CR4-CR5 domain of mammalian telomerase RNA.

Telomerase is an enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The telomerase ribonucleoprotein complex consists of two essential components, a reverse transcriptase and an RNA molecule that provides the template for telomeric repeat synthesis. A common secondary structure of vertebrate telomerase RNA has been proposed based on a phylogenetic comparative analysis of 35 sequences. Here we report the identification of an additional essential base-paired region in the CR4-CR5 domain of mammalian telomerase RNA, termed P6.1. Mouse telomerase RNAs with mutations that disrupted base pairings in the P6.1 helix were unable to reconstitute telomerase activity in vivo. In contrast, an RNA mutant with compensatory mutations that restored base pairings in the P6.1 helix restored telomerase activity. In an in vitro reconstitution system stable base pairing of the P6.1 stem was required for the RNA-protein interaction between the CR4-CR5 domain and the telomerase reverse transcriptase (TERT) protein. Interestingly, two RNA mutations, one that extends the P6.1 stem and one that alters the conserved nucleotides of the L6.1 loop, allowed RNA-protein binding but significantly impaired telomerase activity. These data establish the presence of the P6.1 stem-loop and its importance for the assembly and enzymatic activity of the mammalian telomerase complex.

Animals↗

PubMed2005

Correlated evolution of maternally derived yolk testosterone and early developmental traits in passerine birds.

Recent studies on hormone-mediated maternal effects in birds have highlighted the influence of variable maternal yolk androgen concentration on offspring phenotype, particularly in terms of early development. If genetic differences between laying females regulate variation in yolk hormone concentration, then this physiological maternal effect is an indirect genetic effect which can provide a basis for the co-evolution of maternal and offspring phenotypes. Thus, we investigated the evolutionary associations between maternally derived yolk testosterone (T) and early developmental traits in passerine birds via a comparative, phylogenetic analysis. Our results from species-correlation and independent contrasts analyses provide convergent evidence for the correlated evolution of maternal yolk T concentration and length of the prenatal developmental period in passerines. Here, we show these traits are significantly negatively associated (species-correlation: p<0.001, r2=0.85; independent contrasts: p=0.005). Our results highlight the need for more studies investigating the role of yolk hormones in evolutionary processes concerning maternal effects.

Animals↗

PubMed1996

Phylogenesis of relapsing fever Borrelia spp.

The phylogenetic relationships of 20 relapsing fever (RF) Borrelia spp. were estimated on the basis of the sequences of rrs genes. Complete sequences were aligned and compared with previously published sequences, and the similarity values were found to be 97.7 to 99.9%. Phylogenetic trees were constructed by using the three neighbor-joining, maximum-parsimony, and maximum-likelihood methods. The results of the comparative phylogenetic analysis divided the RF Borrelia spp. into three major clusters. One cluster included Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis, and Borrelia hispanica. Another cluster comprised tow main branches with Borrelia coriaceae, Borrelia lonestari, and Borrelia miyamotoi on one side and Borrelia parkeri, Borrelia turicatae, and Borrelia hermsii on the other side. Borrelia anserina constituted the third cluster. The phylogenetic position of Borrelia persica was more uncertain. These results suggested that the taxonomy of these spirochetes should be revised. To overcome the problems of culturing the spirochetes, RF Borrelia primers were defined. Following PCR amplification of the rrs gene, restriction length fragment polymorphism could be used to distinguish between RF Borrelia strains.

Animals↗

PubMed1998

Delimiting the genus Staphylococcus through description of Macrococcus caseolyticus gen. nov., comb. nov. and Macrococcus equipercicus sp. nov., and Macrococcus bovicus sp. no. and Macrococcus carouselicus sp. nov.

Four species of the newly proposed genus Macrococcus, namely macrococcus caseolyticus gen. nov., comb. nov. (formerly Staphylococcus caseolyticus Schleifer, Kilpper-Bälz, Fischer, Faller and Endl 1982, 19VP), Macrococcus equipercicus sp. nov., Macrococcus bovicus sp. nov. Macrococcus carouselicus sp. nov., are described on the basis of a phylogenetic analysis comparing 16S rRNA sequences, DNA-DNA liquid hybridization, DNA base composition, normalized ribotype patterns, macrorestriction pattern analysis and estimation of genome size using PFGE, cell wall composition, phenotypic characteristics and plasmid profiles. Compared with their closet relatives, members of the genus Staphylococcus, these organisms demonstrated significantly lower 16S rRNA sequence similarities (93.4-95.3%), higher DNA G+C content (38-45 mol%), absence of cell wall teichoic acids (with the possible exception of M. caseolyticus), unique ribotype pattern types and macrorestriction patterns, smaller genome size (approx. 1500-1800 kb) and generally larger Gram-stained cell size (1.1-2.5% microns in diameter). Macrococci can be distinguished from most species of staphylococci (except Staphylococcus sciuri, Staphylococcus vitulus and Staphylococcus lentus) by thier oxidase activity. The four Macrococcus species can be distinguished from one another on the basis of DNA-DNA hybridization, ribotype pattern types, macrorestriction patterns and their phenotypic properties, including colony morphology, cell morphology, haemolysins, Staphy Latex agglutination, acid production from a variety of carbohydrates, acetoin production, nitrate reduction, aesculin hydrolysis, and DNase and urease activities. The type species is M. equipercicus. The type strains of M. equipercicus, M. caseolyticus, M. bovicus and M. carouselicus are ATTCC 51831T (= DD 9350T) ATCC 13548T (= TDD 4508T) (Schleifer et al. 1982, ATCC 51825T (= DD 4516T) and ATCC 51828T (= DD 9348), respectively.

Animals↗

PubMed2001

Herbaspirillum frisingense sp. nov., a new nitrogen-fixing bacterial species that occurs in C4-fibre plants.

The enrichment of nitrogen-fixing bacteria from the C4-fibre plants, Spartina pectinata, Miscanthus sinensis, Miscanthus sacchariflorus and Pennisetum purpureum, with nitrogen-free semi-solid media led to the isolation of Herbaspirillum-like strains among other diazotrophic bacteria. On the basis of physiological properties, phylogenetic analysis comparing 16S rDNA sequences and DNA-DNA hybridization experiments of chromosomal DNA the new isolates could be grouped together in a new species with the proposed name Herbaspirillum frisingense sp. nov. Morphological characteristics, such as cell size and shape, colony appearance, motility and flagellation are largely identical to the known species Herbaspirillum rubrisubalbicans and Herbaspirillum seropedicae. On the basis of utilization of adipate (-), N-acetyl-D-glucosamine (+), meso-erythritol (-), L-rhamnose (-) and meso-inositol (-) Herbaspirillum frisingense sp. nov. can be distinguished from other known Herbaspirillum spp. Nitrogen-fixing capability was examined by PCR amplification of the nifD gene and an acetylene reduction assay, and was found with all isolates tested. 16S rDNA sequence similarity to the other Herbaspirillum spp. is 98.5-99.1%. In genomic DNA-DNA hybridization experiments Herbaspirillum frisingense sp. nov. forms a homogeneous group with 70-100+/-10% similarity, clearly distinct from Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans with 1-34% similarity. 16S rRNA-targeted oligonucleotide probes, specific for the whole genus Herbaspirillum and for three Herbaspirillum species were designed and are suitable for fluorescence in situ hybridization. The DNA G+C content of Herbaspirillum frisingense sp. nov. is 63+/-2 mol%, in agreement with the values of 61-65% for the genus. PCR fingerprinting exhibits a consistent pattern for groups of strains isolated from the same plant, suggesting a low genomic diversity among bacteria inhabiting C4-gramineous plant tissues. Low genetic DNA diversity seems to be common between probable endophytic bacterial isolates of the same taxon. The type strain of Herbaspirillum frisingense sp. nov. is GSF30T (= DSM 13128T).

Base Composition↗

PubMed1998

Nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus, the closterovirus type member.

The entire genome of grapevine leafroll-associated closterovirus-2 (GLRaV-2), except the exact 5' terminus, was cloned and sequenced. The sequence encompasses nine open reading frames (ORFs) which include, in the 5' to 3' direction, an incomplete ORF1a encoding a putative viral polyprotein and eight ORFs that encode proteins of 52 kDa (ORF1b), 6 kDa (ORF2), 65 kDa (ORF3), 63 kDa (ORF4), 25 kDa (ORF5), 22 kDa (ORF6), 19 kDa (ORF7) and 24 kDa (ORF8) respectively, and 216 nucleotides of the 3' untranslated region. An incomplete ORF1a potentially encoded a large polyprotein containing the conserved domains characteristic of a papain-like protease, methyltransferase and helicase. ORF1b potentially encoded a putative RNA-dependent RNA polymerase. The expression of ORF1b may be via a +1 ribosomal frameshift mechanism, similar to other closteroviruses. A unique gene array, which is conserved in other closteroviruses, was also identified in GLRaV-2; it includes genes encoding a 6 kDa small hydrophobic protein, 65 kDa heat shock protein 70, 63 kDa protein of function unknown, 25 kDa coat protein duplicate and 22 kDa coat protein. Identification of ORF6 (22 kDa) as the coat protein gene was further confirmed by in vivo expression in E. coli and immunoblotting. Phylogenetic analysis comparing different genes of GLRaV-2 with those of other closteroviruses demonstrated a close relationship with beet yellows virus (BYV), beet yellow stunt virus and citrus tristeza virus. GLRaV-2 is the only closterovirus, so far, that matches the genome organization of the type member of the group, BYV, and thus can be unambiguously classified as a definitive member of the genus Closterovirus.

Amino Acid Sequence↗

PubMed2006

Three novel species of the genus Catellatospora, Catellatospora chokoriensis sp. nov., Catellatospora coxensis sp. nov. and Catellatospora bangladeshensis sp. nov., and transfer of Catellatospora citrea subsp. methionotrophica Asano and Kawamoto 1988 to Catellatospora methionotrophica sp. nov., comb. nov.

Three Gram-positive, aerobic, non-motile, mesophilic strains, designated 2-25(1)T, 2-29(17)T and 2-70(23)T, were isolated from sandy soil from Chokoria, Cox's Bazar, Bangladesh. The organisms produce short chains of non-motile spores that emerge singly or in tufts from vegetative hyphae on the surface of agar media. A comparative phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates formed a distinct clade within the evolutionary radiation of the family Micromonosporaceae and clustered with members of the genus Catellatospora. The nearest neighbours were Catellatospora citrea subsp. citrea and C. citrea subsp. methionotrophica. Chemotaxonomic data, such as the presence of meso- and 3-hydroxy-diaminopimelic acids, N-glycolyl type muramic acid, arabinose and xylose and glucose in whole-cell hydrolysates, phosphatidylethanolamine as a diagnostic phospholipid, a tetrahydrogenated menaquinone with 9 isoprene units as a major menaquinone and fatty acid profiles predominated by iso-branched hexadecanoic acid and iso-branched pentadecanoic acid, supported the affiliation of the novel isolates to the genus Catellatospora. The results of DNA-DNA hybridization and physiological and biochemical tests allowed the novel isolates to be differentiated genotypically and phenotypically from the three recognized Catellatospora species. The three isolates therefore represent novel species for which the names Catellatospora chokoriensis sp. nov. [type strain 2-25(1)T=JCM 12950T=DSM 44900T], Catellatospora coxensis sp. nov. [type strain 2-29(17)T=JCM 12951T=DSM 44901T] and Catellatospora bangladeshensis sp. nov. [type strain 2-70(23)T=JCM 12949T=DSM 44899T], are proposed. DNA-DNA hybridization tests with C. citrea subsp. citrea and C. citrea subsp. methionotrophica, in combination with chemotaxonomic and physiological data, demonstrated that C. citrea subsp. methionotrophica should be elevated to a separate species for which the name Catellatospora methionotrophica sp. nov., comb. nov. is proposed (type strain JCM 7543T=DSM 44098T).

Bacterial Typing Techniques↗

PubMed2006

Characterization of clinical isolates previously identified as Oerskovia turbata: proposal of Cellulosimicrobium funkei sp. nov. and emended description of the genus Cellulosimicrobium.

Taxonomic studies were performed on 13 clinical isolates (ten of which were epidemiologically related) that had been previously identified as Oerskovia turbata. Comparative phylogenetic analysis, based on 16S rRNA gene sequences, indicated that the isolates are closely related to Cellulosimicrobium cellulans with sequence similarity values ranging from 99.5 to 99.8 %. Chemotaxonomic results (fatty acid profiles and menaquinones) supported the inclusion of these isolates in the genus Cellulosimicrobium. The DNA G+C content was 74.5 mol%. The results of DNA-DNA reassociation, whole-cell sugars (with galactose as the characteristic whole sugar) and phenotypic properties, including antimicrobial resistance, indicated that these isolates are representatives of a novel species of the genus Cellulosimicrobium. The name Cellulosimicrobium funkei sp. nov. is proposed for the novel strains, with strain W6122T (=ATCC BAA-886T = DSM 16025T = CCUG 50705T) as the type strain. The definition of this novel Cellulosimicrobium species will assist in the understanding of the epidemiology and clinical significance of these micro-organisms.

Actinomycetales Infections↗

PubMed2005

Evidence from nature: interspecies spread of heron hepatitis B viruses.

Heron hepatitis B viruses (HHBVs) in three subspecies of free-living great blue herons (Ardea herodias) from Florida, USA, were identified and characterized. Eight of 13 samples were positive in all assays used, whereas sera from egrets, which are also members of the family Ardeidae, were negative in the same assays. Comparative phylogenetic analysis of viral DNA sequences from the preS/S region of previously reported and novel HHBV strains isolated from captive grey herons (Germany) and free-ranging great blue herons (USA), respectively, revealed a strong conservation (95 % sequence similarity) with two separate clusters, implying a common ancestor of all strains. Our data demonstrate for the first time that different subspecies of herons are infected by HHBV and that these infections exist in non-captive birds. Phylogenetic analysis and the fact that the different heron species are geographically isolated populations suggest that lateral transmission, virus adaptation and environmental factors all play a role in HHBV spreading and evolution.

Animals↗

PubMed2002

Evidence for historical introgression along a contact zone between two species of char (Pisces: Salmonidae) in northwestern North America.

Phylogeographic analyses can yield valuable insights into the geographic and historical contexts of contact and hybridization between taxa. Two species of char (Salmonidae), Dolly Varden (Salvelinus malma) and bull trout (S. confluentus) have largely parapatric distributions in watersheds of northwestern North America. They are, however, sympatric in several localities and hybridization and some introgression occurs across a broad area of contact. We conducted a comparative phylogenetic analysis of Dolly Varden and bull trout to gain a historical perspective of hybridization between these species and to test for footprints of historical introgression. We resolved two major Dolly Varden mitochondrial DNA (mtDNA) clades (with 1.4-2.2% sequence divergence between haplotypes) that had different geographical distributions. Clade N is distributed across most of the range of Dolly Varden, from southern British Columbia through to the Kuril Islands in Asia. Clade S had a much more limited distribution, from Washington state, at the southern limit of the Dolly Varden range, to the middle of Vancouver Island. The distribution and inferred ages of the mtDNA clades suggested that Dolly Varden survived the Wisconsinan glaciation in a previously unsuspected refuge south of the ice sheet, and that Dolly Varden and bull trout were probably in continuous contact over most of the last 100,000 years. When bull trout were included in the phylogenetic analysis, however, the mtDNA of neither species was monophyletic: Clade S Dolly Varden clustered within the bull trout mtDNA clade. This pattern was discordant with two nuclear phylogenies produced (growth hormone 2 and rRNA internal transcribed sequence 1), in which Dolly Varden and bull trout were reciprocally monophyletic. This discordance between mtDNA- and nDNA-based phylogenies indicates that historical introgression of bull trout mtDNA into Dolly Varden occurred. Percent sequence divergence within these introgressed Dolly Varden (clade S) was 0.2-0.6%, implying that the introgression occurred prior to the most recent glaciation. Our analysis and other evidence of contact between divergent lineages in northwestern North America strongly suggests that the area may be the site of previously unsuspected suture zones of aquatic biotas.

Animals↗

PubMed2005

The influence of mating system on the intensity of parent-offspring conflict in primates.

An evolutionary conflict of interest exists between parents and their offspring over the partitioning of parental investment (PI) among siblings. When the direct fitness benefits to offspring of increased PI, outweigh the inclusive fitness costs from lost future sibling fitness, selection should favour the evolution of offspring selfishness over altruism. In theory, this conflict is heightened when females are not strictly monogamous, as current offspring should be less altruistic towards future half-siblings than they would be towards full-siblings. Using data collected on foetal growth rate (representing prenatal PI) in primates, I test the prediction from theory that the resolution of the parent-offspring conflict will be closer to the offspring's evolutionary optima in polyandrous species than in more monandrous species. Using phylogenetic comparative analysis, and controlling for allometry, I show that offspring are able to obtain more PI when the probability of future full-siblings decreases, and that this is most pronounced in taxa where there is the opportunity for direct foetal access to the maternal bloodstream. These results support the hypothesis that the resolution of prenatal PI conflict is influenced by both a species' mating system and by its placental structure.

Animals↗

PubMedDate not supplied

Molecular phylogeny and evolution of mosquito parasitic Microsporidia (Microsporidia: Amblyosporidae).

Amblyospora species and other aquatic Microsporidia were isolated from mosquitoes, black flies, and copepods and the small subunit ribosomal RNA gene was sequenced. Comparative phylogenetic analysis showed a correspondence between the mosquito host genera and their Amblyspora parasite species. There is a clade of Amblyospora species that infect the Culex host group and a clade of Amblyospora that infect the Aedes/Ochlerotatus group of mosquitoes. Parathelohania species, which infect Anopheles mosquitoes, may be the sister group to the Amblyospora in the same way that the Anopheles mosquitoes are thought to be the sister group to the Culex and Aedes mosquitoes. In addition, by sequence analysis of small subunit rDNA from spores, we identified the alternate copepod host for four species of Amblyospora. Amblyospora species are specific for their primary (mosquito) host and each of these mosquito species serves as host for only one Amblyospora species. On the other hand, a single species of copepod can serve as an intermediate host to several Amblyospora species and some Amblyospora species may be found in more than one copepod host. Intrapredatorus barri, a species within a monotypic genus with Amblyospora-like characteristics, falls well within the Amblyospora clade. The genera Edhazardia and Culicospora, which do not have functional meiospores and do not require an intermediate host, but which do have a lanceolate spore type which is ultrastructurally very similar to the Amblyospora spore type found in the copepod, cluster among the Amblyospora species. In the future, the genus Amblyospora may be redefined to include species without obligate intermediate hosts. Hazardia, Berwaldia, Larssonia, Trichotuzetia, and Gurleya are members of a sister group to the Amblyospora clades infecting mosquitoes, and may be representatives of a large group of aquatic parasites.

Animals↗

PubMed2003

P-type ATPase superfamily: evidence for critical roles for kingdom evolution.

The P-type ATPase has become a protein superfamily. On the basis of sequence similarities, the phylogenetic analyses, and substrate specificities, this superfamily can be classified into 5 families and 11 subfamilies. A comparative phylogenetic analysis demonstrates the relationship between the molecular evolution of these subfamilies and the establishment of the kingdoms of living things.

Adenosine Triphosphatases↗

PubMed1999

Isolation of West Nile virus from mosquitoes, crows, and a Cooper's hawk in Connecticut.

West Nile (WN) virus, a mosquito-transmitted virus native to Africa, Asia, and Europe, was isolated from two species of mosquitoes, Culex pipiens and Aedes vexans, and from brain tissues of 28 American crows, Corvus brachyrhynchos, and one Cooper's hawk, Accipiter cooperii, in Connecticut. A portion of the genome of virus isolates from four different hosts was sequenced and analyzed by comparative phylogenetic analysis. Our isolates from Connecticut were similar to one another and most closely related to two WN isolates from Romania (2.8 and 3.6 percent difference). If established in North America, WN virus will likely have severe effects on human health and on the health of populations of birds.

Aedes↗

PubMed1999

Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor.

The microbial diversity of a deteriorated biological phosphorus removal reactor was investigated by methods not requiring direct cultivation. The reactor was fed with media containing acetate and high levels of phosphate (P/C weight ratio, 8:100) but failed to completely remove phosphate in the effluent and showed very limited biological phosphorus removal activity. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA was used to investigate the bacterial diversity. Up to 11 DGGE bands representing at least 11 different sequence types were observed; DNA from the 6 most dominant of these bands was further isolated and sequenced. Comparative phylogenetic analysis of the partial 16S rRNA sequences suggested that one sequence type was affiliated with the alpha subclass of the Proteobacteria, one was associated with the Legionella group of the gamma subclass of the Proteobacteria, and the remaining four formed a novel group of the gamma subclass of the Proteobacteria with no close relationship to any previously described species. The novel group represented approximately 75% of the PCR-amplified DNA, based on the DGGE band intensities. Two oligonucleotide rRNA probes for this novel group were designed and used in a whole-cell hybridization analysis to investigate the abundance of this novel group in situ. The bacteria were coccoid and 3 to 4 microm in diameter and represented approximately 35% of the total population, suggesting a relatively close agreement with the results obtained by the PCR-based DGGE method. Further, based on electron microscopy and standard staining microscopic analysis, this novel group was able to accumulate granule inclusions, possibly consisting of polyhydroxyalkanoate, inside the cells.

Bioreactors↗

PubMed1994

Phylogenetic and molecular characterization of a 23S rRNA gene positions the genus Campylobacter in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in Campylobacter spp.

The nucleotide sequence of a 23S rRNA gene of Campylobacter coli VC167 was determined. The primary sequence of the C. coli 23S rRNA was deduced, and a secondary-structure model was constructed. Comparison with Escherichia coli 23S rRNA showed a major difference in the C. coli rRNA at approximately position 1170 (E. coli numbering) in the form of an extra sequence block approximately 147 bp long. PCR analysis of 31 other strains of C. coli and C. jejuni showed that 69% carried a transcribed spacer of either ca. 147 or ca. 37 bp. Comparison of all sequenced Campylobacter transcribed spacers showed that the Campylobacter inserts were related in sequence and percent G+C content. All Campylobacter strains carrying transcribed spacers in their 23S rRNA genes produced fragmented 23S rRNAs. Other strains which produced unfragmented 23S rRNAs did not appear to carry transcribed spacers at this position in their 23S rRNA genes. At the 1850 region (E. coli numbering), Campylobacter 23S rRNA displayed a base pairing signature most like that of the beta and gamma subdivisions of the class Proteobacteria, but in the 270 region, Campylobacter 23S rRNA displayed a helix signature which distinguished it from the alpha, beta, and gamma subdivisions. Phylogenetic analysis comparing C. coli VC167 23S rRNA and a C. jejuni TGH9011 (ATCC 43431) 23S rRNA with 53 other completely sequenced (eu)bacterial 23S rRNAs showed that the two campylobacters form a sister group to the alpha, beta, and gamma proteobacterial 23S rRNAs, a positioning consistent with the idea that the genus Campylobacter belongs to the epsilon subdivision of the class Proteobacteria.

Bacteria↗

PubMed2006

New Staphylococcus aureus genotyping method based on exotoxin (set) genes.

A variety of methods for genotyping Staphylococcus aureus isolates exists: the two most widely used methods are pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Here, we describe a sequence-based genotyping method based on genes encoding S. aureus superantigen-like proteins, which belong to a family of exotoxins called staphylococcal exotoxins. The sequences of PCR-amplified internal fragments of three different set genes (set2, set5, and set7) of 61 well-characterized clinical methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates and reference strains were compared. Phylogenetic analysis was performed based on single-nucleotide polymorphisms (SNP). The SNP dendrograms of the set gene sequences differentiated the 61 isolates into 22 distinct subgroups, designated exotoxin sequence types (ETST), while the standard seven-gene MLST profiles differentiated the same 61 isolates into 19 subgroups. Of the 19 different MLST subgroups, 16 corresponded to 16 distinct ETST groups. However, three MLST subgroups, ST1, ST30, and ST36, were each further separated into more than one ETST subgroup. The exotoxin-based genotyping method was able to discriminate MRSA and MSSA isolates according to their specific epidemiological characteristics. This SNP analysis of the three set genes is thus equally or more discriminatory than the seven-gene MLST method, providing a good alternative typing tool for a laboratory that has sequencing capability.

Bacterial Proteins↗