A novel taxonomic marker that discriminates between morphologically complex actinomycetes

Abstract

In the era when large whole genome bacterial datasets are generated routinely, rapid and accurate molecular systematics is becoming increasingly important. However, 16S ribosomal RNA sequencing does not always offer sufficient resolution to discriminate between closely related genera. The SsgA-like proteins are developmental regulatory proteins in sporulating actinomycetes, whereby SsgB actively recruits FtsZ during sporulation-specific cell division. Here, we present a novel method to classify actinomycetes, based on the extraordinary way the SsgA and SsgB proteins are conserved. The almost complete conservation of the SsgB amino acid (aa) sequence between members of the same genus and its high divergence between even closely related genera provides high-quality data for the classification of morphologically complex actinomycetes. Our analysis validates Kitasatospora as a sister genus to Streptomyces in the family Streptomycetaceae and suggests that Micromonospora, Salinispora and Verrucosispora may represent different clades of the same genus. It is also apparent that the aa sequence of SsgA is an accurate determinant for the ability of streptomycetes to produce submerged spores, dividing the phylogenetic tree of streptomycetes into liquid-culture sporulation and no liquid-culture sporulation branches. A new phylogenetic tree of industrially relevant actinomycetes is presented and compared with that based on 16S rRNA sequences.

Keywords: Streptomyces; cell division; genome sequencing; systematics.

Figure 1.

Maximum-likelihood tree based on the alignment of the 16S rRNA genes of morphologically complex actinomycetes. For input sequences and their accession numbers, see the electronic supplementary material, data file S1.

Figure 2.

Maximum-likelihood tree based on the alignment of RpoB proteins from a range of morphologically complex actinomycetes. For input sequences and their accession numbers, see the electronic supplementary material, data file S2.

Figure 3.

Maximum-likelihood tree based on the alignment of SsgB proteins from a range of morphologically complex actinomycetes. For input sequences and their accession numbers, see the electronic supplementary material, data file S3.

Figure 4.

Alignment of SsgA orthologues. Only those SsgA protein sequences have been used as input that are derived from species with known phenotype in submerged cultures. For shading, at least 60% of the aligned proteins should share the same or similar aa residues. Identical residues shaded black, similar residues shaded grey. Residues highlighted with an asterisk above the alignment, are conserved within—but different between—the ‘LSp’ and ‘NLSp’ branches in figure 5 and function as identifiers for the ability of a certain Streptomyces species to sporulate in submerged culture. Sequences were labelled by their strain of origin, for sequence labels see §4.2. For input sequences and their accession numbers, see the electronic supplementary material, data file S4.

Figure 5.

Phylogenetic tree of SsgA protein and 16S rRNA sequences in streptomycetes. Phylogenetic trees are shown for SsgA (a) and 16S rRNA (b) from 33 Streptomyces species (see §3). Two major branches of SsgA proteins are indicated, namely SsgA orthologues (called type I) from strains that produce typical mycelial clumps and do not produce submerged spores (NLSp branch; indicated with open circles), and SsgA orthologues (called type II) from strains that can produce spores in liquid-culture (LSp branch; closed circles). As an exception, NLSp species S. avermitilis (indicated with a star in figure 5a) carries SsgB variant T128. 16S rRNA-based classification incorrectly positions S. granaticolor in the NLSp branch and Streptomyces species ATCC 3309 in the LSp branch, while the separation between the two subclasses is also far less obvious as highlighted among others by the unresolved position of S. scabies in the 16S rRNA tree. For sequence labels, see §4.2. For input sequences and their accession numbers, see the electronic supplementary material, data file S4.

Figure 5.

Phylogenetic tree of SsgA protein and 16S rRNA sequences in streptomycetes. Phylogenetic trees are shown for SsgA (a) and 16S rRNA (b) from 33 Streptomyces species (see §3). Two major branches of SsgA proteins are indicated, namely SsgA orthologues (called type I) from strains that produce typical mycelial clumps and do not produce submerged spores (NLSp branch; indicated with open circles), and SsgA orthologues (called type II) from strains that can produce spores in liquid-culture (LSp branch; closed circles). As an exception, NLSp species S. avermitilis (indicated with a star in figure 5a) carries SsgB variant T128. 16S rRNA-based classification incorrectly positions S. granaticolor in the NLSp branch and Streptomyces species ATCC 3309 in the LSp branch, while the separation between the two subclasses is also far less obvious as highlighted among others by the unresolved position of S. scabies in the 16S rRNA tree. For sequence labels, see §4.2. For input sequences and their accession numbers, see the electronic supplementary material, data file S4.