A novel taxonomic marker that discriminates between morphologically complex actinomycetes

In the era where large whole genome bacterial data sets 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 (SALPs) are developmental regulatory proteins in sporulating actinomycete, 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 sequence between members of the same genus, and its high divergence even between 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 amino-acid sequence of SsgA is an accurate determinant for the ability of streptomycetes to produce submerged spores, dividing the phylogenetic tree of streptomycetes into LSp (liquid culture sporulation) and NLSp (no liquid culture sporulation) branches. A new phylogenetic tree of industrially relevant actinomycetes is presented and compared to that based on 16S rRNA sequences

Saccharothrix sp. PAL54, a new chloramphenicol-producing strain isolated from a Saharan soil

An actinomycete strain designated PAL54, producing an antibacterial substance, was isolated from a Saharan soil in Ghardaïa, Algeria. Morphological and chemical studies indicated that this strain belonged to the genus Saccharothrix. Analysis of the 16S rDNA sequence showed a similarity level ranging between 96.9 and 99.2% within Saccharothrix species, with S. longispora DSM 43749T, the most closely related. DNA–DNA hybridization confirmed that strain PAL54 belonged to Saccharothrix longispora. It showed very strong activity against pathogenic Gram-positive and Gram-negative bacteria responsible for nosocomial infections and resistant to multiple antibiotics. Strain PAL54 secreted the antibiotic optimally during mid-stationary and decline phases of growth. One antibacterial compound was isolated from the culture broth and purified by HPLC. The active compound was elucidated by uv-visible and NMR spectroscopy and by mass spectrometry. The results showed that this compound was a D(-)-threo chloramphenicol. This is the first report of chloramphenicol production by a Saccharothrix species

Direct mass spectrometric screening of antibiotics from bacterial surfaces using liquid extraction surface analysis

RATIONALE There is a need to find new antibiotic agents to fight resistant pathogenic bacteria. To search successfully for novel antibiotics from bacteria cultivated under diverse conditions, we need a fast and cost‐effective screening method. METHODS A combination of Liquid Extraction Surface Analysis (LESA), automated chip‐based nanoelectrospray ionization, and high‐resolution mass or tandem mass spectrometry using an Orbitrap XL was tested as the screening platform. Actinobacteria, known to produce well‐recognized thiazolyl peptide antibiotics, were cultivated on a plate of solid medium and the antibiotics were extracted by organic solvent mixtures from the surface of colonies grown on the plate and analyzed using mass spectrometry (MS). RESULTS LESA combined with high‐resolution MS is a powerful tool with which to extract and detect thiazolyl peptide antibiotics from different Actinobacteria. Known antibiotics were correctly detected with high mass accuracy (<4 ppm) and structurally characterized using tandem mass spectra. Our method is the first step toward the development of a novel high‐throughput extraction and identification tool for antibiotics in particular and natural products in general. CONCLUSIONS The method described in this paper is suitable for (1) screening the natural products produced by bacterial colonies on cultivation plates within the first 2 min following extraction and (2) detecting antibiotics at high mass accuracy; the cost is around 2 Euro per sample