Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic contentmust be representative of the total microbial diversity. In this study,we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghumendophytic diversities. Therefore, “SDS-extracted” sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDSbased DNA extraction protocol allows the recovery of the most diverse endophytic communities associated with sorghum tissues and, as such, establishes a reliable basis for future study of endophytic communities.South African National Research Foundation (NRF) Grant No: 71081.http://www.elsevier.com/locate/jmicmeth2016-05-31hb201

Metaviromics of Namib desert salt pans : a novel lineage of haloarchaeal salterproviruses and a rich source of ssDNA viruses

Viral communities of two different salt pans located in the Namib Desert, Hosabes and Eisfeld, were investigated using a combination of multiple displacement amplification of metaviromic DNA and deep sequencing, and provided comprehensive sequence data on both ssDNA and dsDNA viral community structures. Read and contig annotations through online pipelines showed that the salt pans harbored largely unknown viral communities. Through network analysis, we were able to assign a large portion of the unknown reads to a diverse group of ssDNA viruses. Contigs belonging to the subfamily Gokushovirinae were common in both environmental datasets. Analysis of haloarchaeal virus contigs revealed the presence of three contigs distantly related with His1, indicating a possible new lineage of salterproviruses in the Hosabes playa. Based on viral richness and read mapping analyses, the salt pan metaviromes were novel and most closely related to each other while showing a low degree of overlap with other environmental viromes.National Research Foundation (NRF) of South Africa, Claude Leon Foundation and the Vice Chancellor’s Postdoctoral Fellowship program of the University of Pretoria.http://www.mdpi.com/journal/viruseshb201

Identification and characterization of a novel Geobacillus thermoglucosidasius bacteriophage, GVE3

The study of extremophilicphages may reveal new phage families as well as different mechanisms of infection, propagation and lysis to those found in phages from temperate environments. We describe a novel siphovirus, GVE3, that infects the thermophileGeobacillusthermoglucosidasius. The genome size is 141298 bp(G+C 29.6%) making it the largest Geobacillusspp infecting phage known.GVE3 appears to be most closely related to the recently described Bacillus anthracis phage vB_BanS_Tsamsa, rather thanGeobacillus infecting phages described thus far.Tetranucleotide usage deviation analysis supports this relationship, showing that the GVE3 genome sequence correlates best with B. anthracis and Bacillus cereus genome sequences, rather than Geobacillusspp genome sequences.National Research Foundation (NRF) of South Africahttp://link.springer.com/journal/7052016-09-30hb201