FBIP: Molecular signatures to define members of the actinobacterial family Streptosporangiaceae

Study Extent

Global coverage

Sampling

All the actinobacterial type strains were purchased from international culture collections, except the type strain of Nonomuraea candida, which was isolated by us (REFERENCE: Le Roes, M. and Meyers, P. R. (2008) Nonomuraea candida sp. nov., a new species from South African soil. Antonie van Leeuwenhoek; 93: 133-139).

Method steps

1. What will be done DNA sequences will be obtained from the recA, rpoB and relA genes for each type strain in the family treptosporangiaceae and also for several non-type strains. Amino acid sequences will be obtained by in silico translation of the gene sequences. For each protein, the amino acid sequences for all strains in each genus will be aligned and the alignment will be used to define a consensus amino-acid sequence for that protein for each genus (positions with variable amino acids will be designated as X). The resulting consensus amino acid sequences for each gene for the 13 genera will then be aligned and this alignment will be inspected for amino acids that are unique to each genus (genusspecific amino acid indels and amino acid sequences). These unique indels and/or sequences will be designated as signature amino acids for that genus. The identified molecular signatures will serve as amino-acid barcodes for each genus. Furthermore, for each protein, the alignment of consensus amino acid sequences for the 13 genera will also be used to define a consensus sequence for that protein for the family Streptosporangiaceae (i.e. a sequence showing the amino acids common to all strains of all genera in the family and therefore serving as a barcode for that protein for the family). Should any of the chosen genes prove to be unsuitable in distinguishing between genera in the family Streptosporangiaceae, there are several other genes that have been identified in the published literature as being potentially useful in bacterial taxonomy. Possible alternative genes are atpD, trpB and wblA. Method and approach The strains in the family Streptosporangiaceae will be grown under conditions (growth medium and temperature) that favour the production of a large amount of cell mass. Genomic DNA will be isolated from each strain using a well-established method that provides high DNA concentrations. The DNA will be stored at -20°C. PCR primers will be designed that will allow each gene (recA, rpoB and relA) to be amplified in two or more overlapping sections using Taq DNA polymerase. PCR-amplified fragments will be sequenced by Sanger sequencing and the sequences will be assembled into a single consensus sequence for each gene for each strain. Two sequences for each section of each gene will be obtained: one sequence from each of two different amplicons covering that section of the gene, so as to be able to identify and correct any Taqinduced sequencing errors. We will obtain sequences for each gene from each member of every genus in the family. For the multi-species genera (10 genera), we will initially obtain sequences from three to five phylogenetically distinct type strains in the genus (phylogenetic distinctiveness will be determined based on 16S rRNA and gyrB gene trees). This will allow us to assess whether each gene generates phylogenetic trees in which strains from the same genus form a group that is separated from the strains of other genera. We will also look for early indications of amino acid indels and/or signatures that distinguish the genera from each other. If the early results are positive for a particular gene, we will then proceed to obtain the sequences for that gene from all members of the family Streptosporangiaceae. If any gene is shown to have similar sequences between genera, it is unlikely that that gene will be taxonomically useful (as genera cannot be easily distinguished from each other based on sequences of this gene). In this case, we will substitute the unsuitable gene for another gene.

All specimen identified to Species level

1. Streptosporangiaceae
   common name: Bacteria rank: family

Global