Bacteriology
The sigH gene sequence can subspeciate staphylococci

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Abstract

In an evolutionarily conserved gene organization (syntenic region), the sigH gene shares exceptionally low homology among staphylococcal species. We analyzed the “positionally cloned” sigH sequences of 39 staphylococcal species. The topology of the SigH phylogenetic tree was consistent with that of 16S rRNA. Certain clinical isolates were successfully differentiated at the species level with the sigH sequence data set. We propose that the sigH gene is a promising molecular target in genotypic identification because it is highly discriminative in differentiating closely related staphylococcal species.

Introduction

The Staphylococcus genus is currently classified into 39 species (Euzeby, 1997). The precise species identification of staphylococci is important in etiologic and epidemiologic studies. Phenotypic tests are broadly used in today's clinical laboratories, but precise discrimination of the species often requires additional information (Grant et al., 1994, Perl et al., 1994, Renneberg et al., 1995). Several gene sequences such as 16S rRNA (Becker et al., 2004, Fujita et al., 2005, Skow et al., 2005, Takahashi et al., 1999), tuf (Martineau et al., 2001), rpoB (Drancourt & Raoult, 2002, Mellmann et al., 2006), hsp60 (Goh et al., 1997, Kwok & Chow, 2003), gap (Yugueros et al., 2000), femA (Vannuffel et al., 1999), and sodA (Giammarinaro et al., 2005, Poyart et al., 2001, Sivadon et al., 2004) have been used in the identification of staphylococcal species. However, highly conserved genes are not suitable for the discrimination of closely related species.

The sigH gene encodes a sigma factor that constitutes a large alternative sigma factor group in Firmicutes, and it is located within an evolutionarily conserved gene organization (Morikawa et al., 2003). In sporulating bacteria such as Bacillus subtilis and Clostridium perfringens, SigH regulates the initiation of sporulation and is highly conserved even among the distinct genera. In contrast, SigH in nonsporulating bacteria such as Staphylococcus and Streptococcus shares low homology among species.

In this study, we analyzed the sigH sequences of 39 staphylococcal species, aiming to show that the sigH gene is a promising molecular target for discriminating between closely related species. In addition, bioinformatic analysis revealed that there are similar gene clusters with high local diversity, suggesting that such syntenic regions may be promising molecular targets for species identification.

Section snippets

Bacterial strains

Staphylococcus strains used in this study are shown in Table 1. Clinical isolates of Staphylococcus saprophyticus candidates were obtained as described previously (Higashide et al., 2006). Chromosomal DNA was extracted using conventional methods.

Polymerase chain reaction amplification of the sigH locus and sequencing analysis

Polymerase chain reaction (PCR) primers (Table 1) were designed for the conserved region outside the sigH coding sequence (Fig. 1). The sigH locus was amplified by PCR using Ex-taq polymerase (Takara, Otsu, Japan). The PCR products were purified by

The sigH gene is located between conserved genes

The sigH gene has a low homology among species but is embedded in a conserved gene cluster (Fig. 1) (Morikawa et al., 2003). The genes adjacent to sigH in Staphylococcus epidermidis, Staphylococcus haemolyticus, and S. saprophyticus share more than 70% identity with those in Staphylococcus aureus, whereas the sigH gene shares less than 40% identity. We supposed that such a local diversity would be useful for identifying species; the high divergence of the target gene is necessary to

Discussion

We established the sigH sequence data set of all 39 staphylococcal species. Its high divergence is useful for discriminating between closely related species that cannot be assigned by conventional phenotypic testing or by using more highly conserved genes. Among the genes tested so far, the 1 most successfully used for discriminating between closely related species is sodA (Poyart et al., 2001, Sivadon et al., 2004, Sivadon et al., 2005). It was used to design hybridization probes and enabled

Acknowledgment

The authors thank Mr. Masato Higashide (Kotobiken Medical Laboratories, Japan) for clinical isolates and Mr. Yu Ohki for DNA sequencing. We are also grateful to Dr. Flaminia Miyamasu for correction of the manuscript. This work was supported by a Grant-in-Aid for Young Scientists of the Japan Society for the Promotion of Science (to K.M.).

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