FBIP: Actinobacterial diversity in selected South African peatlands

Study Extent

South Africa (Western Cape)

Sampling

Site identification and sample collection, Isolation and cultivation of actinobacteria, Identification of actinobacterial isolates, Metagenomic analyses of actinobacterial populations and the occurrence of genes encoding for small laccases

Method steps

1. Task 1: Site identification and sample collection Four sites have been identified: 1) a “young” peatland in the early stages of establishment (located on Springfield farm, Agulhas; fynbos dominated region); 2) a peatland disrupted by farming (located near the Goukou River, Riversdale; palmiet-dominated region); 3) an “old” peatland that due to alien invasion by black wattle has become unstable and has been eroded away by environmental conditions (originally a palmiet-dominated region); and 4) a pristine peatland near Sedgefield (Vankersvelvlei), known to be the only peatland in South Africa to have sphagnum moss associated with it (Grundling & Grobler, 2005). At each site, four samples will be taken at different depths, up to 1 m. A bulk sample (approximately 100g) will also be taken for physicochemical characterisation (pH, C/N ratio, total N, total P, total K; to be conducted at Bemlab, Somerset West). Sampling will be conducted in summer, so as to allow for access to the sites identified, which typically become waterlogged during winter months. The GPS co-ordinates of the sites will be recorded as well as the vegetation types (photographic recording) for later identification/verification by the Kirstenbosch plant identification service. Task 2: Isolation and cultivation of actinobacteria The standard isolation technique will be used for the isolation of actinobacterial strains: 1 g of soil is suspended in 1 ml of sterile distilled water, vortexed and serially diluted in sterile distilled water. The diluted samples are plated onto different media types containing different carbon sources, e.g. Czapek solution agar (sucrose), yeast extract malt extract agar (glucose), and JCM medium 61 (starch), as well as minimal media (water agar with soil extract) and a rich medium (modified phenoxazinone production medium; Le Roes-Hill et al., 2011). All of the media will be supplemented with cycloheximide and nalidixic acid to limit fungal and Gram negative bacterial growth, respectively. Plates will be incubated at 30 degrees Celcius for 21 days to allow slow-growing actinobacteria to become visible on the plates. The samples will also be subjected to a pre-treatment step prior to plating: 1g of soil will be mixed with 1g of calcium carbonate, ground with a pestle and mortar and incubated at 30 degrees Celcius in a moist environment for 10 days. Following this incubation, the samples will be serially diluted as for the standard isolation technique. Actinobacteria will be identified based on their characteristic colony morphology and will be sub-cultured onto fresh agar plates. Pure cultures will be grown in liquid media and stored as glycerol stocks (20%, v/v) at -20 and -80 degrees Celcius. Each strain will be allocated a unique identifying number which will allow them to be linked to a specific isolation site and isolation medium. Task 3: Identification of actinobacterial isolates Well-established techniques are in place for the identification of novel actinobacterial species. This polyphasic approach typically starts with the identification of the isolate through the sequencing of the 16S rRNA gene. The relationship of the isolate to other strains can be determined through the submission of the gene sequence to the online EzTaxon-e server for identification and the phylogenetic analyses can be performed through the use of the freeware MEGA 6 to determine which strains are the most closely related to the isolate(s) of interest. A comparative study of the phenotypic features of the novel strains and their closest neighbours will be performed. In addition, the chemotaxonomic properties of the strains will be determined by the bacterial identification service at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Novel species will be submitted to two international culture collections for verification of identity. Task 4: Metagenomic analyses of actinobacterial populations and the occurrence of genes encoding for small laccases Total metagenomic DNA will be isolated from the peat samples using a soil-DNA isolation kit (PowerSoil®DNA Isolation Kit, MO BIO Laboratories). Actinobacterial 16S rRNA genes will be amplified using universal 16S rRNA gene PCR primers (Cook and Meyers, 2003) and the amplified products used as the template for a second, nested PCR using actinobacterial-specific 16S rRNA gene primers SC-Act-235aS20 and SC-Act-878aA19 (Stach et al., 2003) and/or Com2xf and Ac1186r (Schäfer et al., 2010). The metagenomic DNA samples will also be used for the amplification of actinobacterial small laccases, using the primer set, LacK155F and LacK120R, designed by Fernandes et al. (2014). Actinobacterial-specific 16S rRNA gene amplicons and small laccase (HFamily K1) amplicons will be sequenced through the next generation sequencing service provided by Inqaba Biotech. The sequence data will be processed via their bioinformatics platform.

Bacteria

1. Actinobacteria
   rank: phylum

South Africa (Western Cape)